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ANTICANCER ACTIVITY STUDIES: DIFFERENT MODELS Jesil Mathew. A, MCOPS, Manipal University
US Mortality, 2005 <ul><li>1. Heart Diseases 652,091  26.6 </li></ul><ul><li>  </li></ul><ul><li>2. Cancer 559,312  22.8 <...
2008 Estimated US Cancer Deaths* ONS=Other nervous system. Source: American Cancer Society, 2008. Men 294,120 Women 271,53...
Change in the US Death Rates* by Cause,  1950 & 2005 * Age-adjusted to 2000 US standard population. Sources: 1950 Mortalit...
<ul><li>Neoplasia   is any new or continued cell growth not needed for normal development or replacement of dead and damag...
Evolution of Cancer <ul><li>Human body has  ~10 14  cells from a single egg cell. </li></ul><ul><li>Typical adult cell  ha...
<ul><li>Normal cells </li></ul><ul><li>Have limited cell division </li></ul><ul><li>Undergo apoptosis </li></ul><ul><li>Sh...
<ul><li>Cancer cells </li></ul><ul><li>Have rapid or continuous cell division </li></ul><ul><li>Do not respond to signals ...
Characteristics of cancer <ul><li>They divide - generally relatively fast. </li></ul><ul><li>Often </li></ul><ul><ul><li>H...
Targets of anticancer drugs: conventional  <ul><li>Cell division: </li></ul><ul><ul><li>Pro - valid for all cancers.  </li...
<ul><li>Targeting Signaling and associated molecules </li></ul>Targets of anticancer drugs: novel approaches
There are many ways to die…..
Cell Viability •  Functional assay •  Membrane integrity assay   •  DNA labeling assay •  Morphological assay  •  Reproduc...
The major criteria employed in viability assay
Cell counting
<ul><li>   Membrane integrity assay </li></ul><ul><li>Hemocytometer  Trypan blue assay / Automatic trypan blue method </l...
Membrane integrity assays
Dye exclusion methods <ul><li>Viability assays measure the percentage of a cell suspension that is viable.  </li></ul><ul>...
Trypan blue    A stain which will only  enter across the membranes  of dead/non-viable cells. -  Cause cancer  in lab. an...
<ul><li>The  most common routine method for cell counting  which is efficient and accurate is with the use of a hemocytome...
Volume : 0.1mm 3 1 ml = 1 cm 3  = 1000 mm 3 1 mm 1 mm 0.1 mm
Dead cell
Materials and Equipment <ul><li>   Trypan blue (0.4 g trypan blue in 100 ml physiological saline) -> pass through a 0.22 ...
Methods <ul><li>Clean hemocytometer & coverslip and  wipe with 70% alcohol before use </li></ul><ul><li>Place coverslip  o...
<ul><li>Calculations </li></ul><ul><li>A = Vol. Of cell solution (ml) </li></ul><ul><li>B = Dilution factor in trypan blue...
Example <ul><li>Vol. : Volume </li></ul><ul><li>CS : Cell Solution </li></ul><ul><li>TB : Trypan blue </li></ul>Dilution f...
Automated trypan blue method for optimal cell viability determination www.innovatis.com
Introduction <ul><li>   The trypan blue dye exclusion assay is the most commonly used and accepted method for the  measur...
<ul><li>   The system consist of three functional part: the liquid handling unit, image capture hard ware, and a data pro...
Result view of the image processing Marked viable and dead cells Viable cell Dead cell
Fig. a)  Distribution histogram of the   viable cell diameter  for a human leukemia cell sample. b)  Histogram of compactn...
Vi-CELL  TM  CELL Viability Analyzer
 
Principle Trypan Blue dye Exclusion Methods
Run results
<ul><li>One parameter of cell death is the integrity of the cell membrane. </li></ul><ul><li>It can be measured by cytopla...
<ul><li>   Quantitative value  for the loss of cell viability </li></ul><ul><li>   The activity of LDH can be measured  ...
Requirement of LDH assay <ul><li>Greater process productivity  (e.g. High-cell-density entrapped-culture systems) : diffic...
Pitfalls of LDH assay <ul><li>   The release of  LDH activity  can be related to the  total No. of dead & lysed cells. </...
Reagents and Solutions <ul><li>Buffer  (Tris 81.3 mmol/L ; NaCl 203.3 mmol/L ; pH 7.2)  </li></ul><ul><li>: Dissolve 4.92 ...
<ul><li>   Buffer  is stable at  0-4℃. </li></ul><ul><li>   The NADH solution  is kept at  0-4℃  and must be prepared  f...
Materials and Equipment <ul><li>   NADH solution </li></ul><ul><li>   Pyruvate solution </li></ul><ul><li>   Narrow-ban...
 
<ul><li>The LDH activity is determined by an enzymatic test. </li></ul><ul><li>The first step is the reduction of NAD +  t...
<ul><li>   Ethidium bromide (EtBr)  and  propidium iodide (PI) </li></ul><ul><li>PI is impermeable to intact plasma membr...
<ul><li>   Fluorescein diacetate (FDA)  is a nonpolar ester which passes through plasma membranes and is hydrolyzed by in...
Schematic illustration of the principle of  PI/FDA cell viability assay   Intact cell – PI and FDA is added Fluorescein in...
Example 1 ; Observation of cell death   A group of hepatoma cells exposed to a diffusing wave of digitonin. Intact cells (...
Evaluate viability by  examining the metabolic components that are necessary for cell growth , on the premise that cellula...
Colorimetric assay <ul><li>   Rapid and accurate  assessment of viable cell number </li></ul><ul><li>   Miniaturized int...
<ul><li>   These assay are read at 570 nm (except for the Acid Phosphatase (A.P) assay-wavelength is 405 nm) on a ELISA p...
<ul><li>Introduction </li></ul><ul><li>   This assay is a  sensitive, quantitative and  reliable colorimetric assay  that...
<ul><li>   The amount of formazan produced is directly  proportional to the cell number in range of cell lines. </li></ul...
<ul><li>Materials and equipment </li></ul><ul><li>MTT solution (5  ㎎ / ㎖  in phosphate buffered saline (PBS) pH 7.5), HCl,...
<ul><li>Advantages of MTT assay </li></ul><ul><li>Considered a major advance; used the most prevalent in vitro assay </li>...
<ul><li>Disadvantage of MTT assay </li></ul><ul><li>Production of the MTT product is dependent on the MTT concentration in...
Example; NIH J2 3T3 cell in medium MTT solution elution Absorbance at 540 nm
XTT assay <ul><li>Introduction </li></ul><ul><li>   The assay is based on the cleavage of the yellow tetrazolium salt XTT...
metabolically active cell Water soluble
<ul><li>Materials and equipments </li></ul><ul><li>XTT labeling regent, electron-coupling reagent </li></ul><ul><li>96-wel...
<ul><li>Compare with MTT assay and XTT assay </li></ul>Culture cells in a MTP  for a certain period  of time (37℃) MTT ass...
Example:  MTT and XTT MTT XTT Jenny G., Mark H., Anna J., Inger K., Douglas Mc., Roland M., 2002.  Evaluation of redox ind...
Principle  <ul><li>   Dye elution:  </li></ul><ul><li>Cell up-taken dye was measured colorimetric method after acetic aci...
Procedure <ul><li>   Dye elution   </li></ul><ul><li>①  After  removal of medium, rinse 96 well plates with 100  ㎕ /well ...
Example; Monolayer culture Ref.:Journal of Orthopaedic Resarch19 (2001) ※  OP : Osteoporosis Crystal Violet - 60minutes No...
Example; Microcarrier culture Rabbit oral mucosal cell cultured by microcarrier   Cell counting
Acid phosphatase (AP) assay <ul><li>Introduction </li></ul><ul><li>   The action of this enzyme in many of tissue is to  ...
P-nitrophenyl phosphate + Acid phosphatase    Nitrophenol + HPO 4 -2
<ul><li>Materials and equipment </li></ul><ul><li>Substrate-containing buffer : 10 mM P-nitrophenyl phosphate in 0.1 M sod...
<ul><li>   Principle </li></ul><ul><li>In the presence of cellular metabolism the color of Alamar Blue (ALB) changes from...
Procedure
 
Neutral Red assay ( 3-amino-7dimethyl-2-methyphenazine hydrochloride) <ul><li>   Principle </li></ul><ul><li>- The  incor...
Materials and Equipments <ul><li>   Solution </li></ul><ul><li>①  Neutral red </li></ul><ul><li>4mg/ml stock solution </l...
Procedure <ul><li>①   Resuspend cells of actively growing culture and count cells and accurately allocate appropriate numb...
Principle <ul><ul><li>The rate of DNA synthesis is a reflection of proliferation under many condition.To measure the proli...
Schematic diagram of [ 3 H]-TdR and BrdU
Labeling index with [ 3 H]-thymidine <ul><li>①  Set up the culture at 2x10 4  cells/ml~ 5x10 4  cells/ml in 24 well plates...
DNA synthesis by [ 3 H]-thymidine <ul><li>①  Grow the culture to the desired density. </li></ul><ul><li>②  [ 3 H]-TdR, 40 ...
DNA labeling assay   (using fluorescent probes assay)
Two types of nonviable cells
Two methods:  Enzymatic DNA labeling and DNA-binding dye labeling
Ⅰ . Enzymatic DNA labeling dNTP dUTP Direct Indirect X Fluoresein, FITC, PE etc Biotin DIG Avidin conjugated with fluorese...
Fig. Immunostaining of apoptotic cells (dark brown) by TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling ...
DNA-binding dyes: fluorochrome 1  RNase treatment is required 2  RNase treatment is required because PI could stain double...
Pattern of dye staining according to color and chromatin morphology * late apoptosis   is regarded as the stage of membran...
Fig. In  Hoechst 33258 / PI double staining,  cells with blue intact nuclei were viable cells, whereas those with blue fra...
Fig. DAPI staining of condensed nuclei of apoptotic cells
Acridine orange
<ul><li>   Fluorescent protein biosensors measuring the molecular dynamics of macromolecules, metabolites, and ions in si...
<ul><li>   PI intercalates DNA double strands in dead cells  without regard to cell types, as their membrane lose integri...
Fig. Each part of quadrant statistics was observed under fluorescence microscopy.  Live ECs preferentially expressed the g...
Morphological assay
<ul><li>   Large-scale, morphological changes that occur at the cell surface, or in the  cytoskeleton , can be followed a...
Example;  Morphological feature   (Human skin keratinocyte)   Fig. Morphological feature of (A) normal human skin keratino...
Example;  Morphological feature   (Human skin fibroblasts) Fig. Morphological feature of (A) normal human skin fibroblasts...
Reproductive Assay
<ul><li>Clonogenic Cell: </li></ul><ul><li>   Defined as a cell with the capacity for sustained proliferation </li></ul><...
<ul><li>Colony-forming Efficiency (CFE) </li></ul><ul><li>   The ability to form colonies is used as a measure of </li></...
Determining the PE of an established adherent cell line   <ul><li>Materials and Equipment </li></ul><ul><li>   Cell  grow...
Procedure <ul><li>1. Trypsinize monolayer cultures or use cell suspension  </li></ul><ul><li>cultures and determine the vi...
Example;   Rat keratinocytes (A) (B) (C) (D) : colony ,  : Single cells Colony forming Non-colony forming 48 hr after subc...
Reference <ul><li>1. Cell quantification, module 4B:1, Hemocytometer cell counts and viability studies, 1.1 – 1.5 </li></u...
<ul><li>7.  Maria L. Anthony, Shane N. O. Williams, and Kevin M. Brindle, Nuclear magnetic resonance methods of monitoring...
In-vivo models
Models of transplantable tumour <ul><li>Murine solid tumour models with Daltons lymphomatic cells </li></ul>
Ascites tumor model <ul><li>Normal mouse </li></ul><ul><li>Tumor bearing mouse </li></ul>
 
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Anticancer activity studies

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Anticancer activity studies

  1. 1. ANTICANCER ACTIVITY STUDIES: DIFFERENT MODELS Jesil Mathew. A, MCOPS, Manipal University
  2. 2. US Mortality, 2005 <ul><li>1. Heart Diseases 652,091 26.6 </li></ul><ul><li> </li></ul><ul><li>2. Cancer 559,312 22.8 </li></ul><ul><li>3. Cerebrovascular diseases 143,579 5.9 </li></ul><ul><li> </li></ul><ul><li>4. Chronic lower respiratory diseases 130,933 5.3 </li></ul><ul><li> </li></ul><ul><li>5. Accidents (unintentional injuries) 117,809 4.8 </li></ul><ul><li> </li></ul><ul><li>6. Diabetes mellitus 75,119 3.1 </li></ul><ul><li> </li></ul><ul><li>7. Alzheimer disease 71,599 2.9 </li></ul><ul><li> </li></ul><ul><li>8. Influenza & pneumonia 63,001 2.6 </li></ul><ul><li> </li></ul><ul><li>9. Nephritis* 43,901 1.8 </li></ul><ul><li>10. Septicemia 34,136 1.4 </li></ul>*Includes nephrotic syndrome and nephrosis. Source: US Mortality Data 2005, National Center for Health Statistics, Centers for Disease Control and Prevention, 2008. Rank Cause of Death No. of deaths % of all deaths
  3. 3. 2008 Estimated US Cancer Deaths* ONS=Other nervous system. Source: American Cancer Society, 2008. Men 294,120 Women 271,530 26% Lung & bronchus 15% Breast 9% Colon & rectum 6% Pancreas 6% Ovary 3% Non-Hodgkin lymphoma 3% Leukemia 3% Uterine corpus 2% Liver & intrahepatic bile duct 2% Brain/ONS 25% All other sites Lung & bronchus 31% Prostate 10% Colon & rectum 8% Pancreas 6% Liver & intrahepatic 4% bile duct Leukemia 4% Esophagus 4% Urinary bladder 3% Non-Hodgkin 3% lymphoma Kidney & renal pelvis 3% All other sites 24%
  4. 4. Change in the US Death Rates* by Cause, 1950 & 2005 * Age-adjusted to 2000 US standard population. Sources: 1950 Mortality Data - CDC/NCHS, NVSS, Mortality Revised. 2005 Mortality Data: US Mortality Data 2005, NCHS, Centers for Disease Control and Prevention, 2008. Heart Diseases Cerebrovascular Diseases Influenza & Pneumonia Cancer 1950 2005 Rate Per 100,000
  5. 5. <ul><li>Neoplasia is any new or continued cell growth not needed for normal development or replacement of dead and damaged tissues. </li></ul><ul><li>Tumor & Cancer : In medical language, a neoplasm is often referred as tumor. </li></ul><ul><li>Malignant </li></ul><ul><li>Benign </li></ul><ul><li>Metastasis </li></ul>What is cancer
  6. 6. Evolution of Cancer <ul><li>Human body has ~10 14 cells from a single egg cell. </li></ul><ul><li>Typical adult cell has gone through ~50 divisions. </li></ul><ul><li>Each division is accompanied by 1-10 errors typically - so total around 50-500 mutations per cell. But 90% of mutations will do nothing. </li></ul><ul><li>Additional mutations can occur from UV, radiation, virus, etc. </li></ul><ul><li>Mutations that favor growth of an individual cell over survival of the organism lead to cancer. </li></ul><ul><li>Activation of pro-growth “oncogenes” </li></ul><ul><li>Suppression of anti-growth “tumor suppressor genes” </li></ul><ul><li>Occassionally can get cancer without mutation. </li></ul><ul><li>best example is teratoma - are the result of abnormal development of pluripotent cells: germ cells and embryonal cells . </li></ul>
  7. 7. <ul><li>Normal cells </li></ul><ul><li>Have limited cell division </li></ul><ul><li>Undergo apoptosis </li></ul><ul><li>Show specific morphology </li></ul><ul><li>Have a small nuclear-cytoplasmic ratio </li></ul><ul><li>Perform specific differentiated functions </li></ul><ul><li>Adhere tightly together </li></ul><ul><li>Nonmigratory </li></ul><ul><li>Grow in an orderly and well-regulated manner </li></ul><ul><li>Contact inhibited </li></ul>Normal cells Vs Cancer cells
  8. 8. <ul><li>Cancer cells </li></ul><ul><li>Have rapid or continuous cell division </li></ul><ul><li>Do not respond to signals for apoptosis </li></ul><ul><li>Show anaplastic morphology </li></ul><ul><li>Have a large nuclear-cytoplasmic ratio </li></ul><ul><li>Lose some or all differentiated functions </li></ul><ul><li>Adhere loosely together </li></ul><ul><li>Able to migrate through embryonic cells </li></ul><ul><li>Grow by invasion </li></ul>Normal cells Vs Cancer cells
  9. 9. Characteristics of cancer <ul><li>They divide - generally relatively fast. </li></ul><ul><li>Often </li></ul><ul><ul><li>Have chromosomes are fused/split, with some deleted and some duplicated. </li></ul></ul><ul><ul><li>Have poor DNA repair mechanisms/high mutation rates. </li></ul></ul><ul><ul><li>More fragile than normal cells. </li></ul></ul><ul><ul><li>Retain characteristics of normal cells they descended from </li></ul></ul><ul><ul><li>Many breast cancers require estrogen and/or progesterone to proliferate </li></ul></ul>
  10. 10. Targets of anticancer drugs: conventional <ul><li>Cell division: </li></ul><ul><ul><li>Pro - valid for all cancers. </li></ul></ul><ul><ul><li>Con - bad for bone marrow, gut, hair, etc. </li></ul></ul><ul><li>Recruitment of blood vessels: </li></ul><ul><ul><li>Pro - prevents tumors from getting large. May make other chemotherapy more effective. </li></ul></ul><ul><ul><li>Con - small tumors can still cause harm. Long term effects may reduce overall circulation. </li></ul></ul><ul><li>DNA repair: </li></ul><ul><ul><li>Pro - works on many cancers </li></ul></ul><ul><ul><li>Con - not all cancers have repair defects. Stresses other cells. </li></ul></ul><ul><li>Hormones: </li></ul><ul><ul><li>Pro - relatively non-toxic. (Don’t *need* estrogen). </li></ul></ul><ul><ul><li>Con - doesn’t work on all cancers. </li></ul></ul>
  11. 11. <ul><li>Targeting Signaling and associated molecules </li></ul>Targets of anticancer drugs: novel approaches
  12. 12. There are many ways to die…..
  13. 13. Cell Viability • Functional assay • Membrane integrity assay • DNA labeling assay • Morphological assay • Reproductive assay
  14. 14. The major criteria employed in viability assay
  15. 15. Cell counting
  16. 16. <ul><li> Membrane integrity assay </li></ul><ul><li>Hemocytometer Trypan blue assay / Automatic trypan blue method </li></ul><ul><li>LDH(lactate dehydrogenase) leakage </li></ul><ul><li>Fluorescent dyes </li></ul><ul><li> Functional Assay </li></ul><ul><li>MTT / XTT assay </li></ul><ul><li>Crystal violet / Acid phosphatase(AP) assay </li></ul><ul><li>Alamar Blue oxidation-reduction assay / Neutral red assay </li></ul><ul><li>[3H]-thymidin and BrdU incorporation </li></ul><ul><li> DNA Assay </li></ul><ul><li>Enzymatic DNA labeling / DNA-binding dye labeling </li></ul><ul><li> Morphological assay </li></ul><ul><li> Reproductive Assay </li></ul><ul><li>Colony-forming efficiency </li></ul><ul><li> Laser scanning confocal microscopy </li></ul><ul><li> Nuclear magnetic resonance methods </li></ul>
  17. 17. Membrane integrity assays
  18. 18. Dye exclusion methods <ul><li>Viability assays measure the percentage of a cell suspension that is viable. </li></ul><ul><li>This is generally accomplished by a dye exclusion stain, where cells with an intact membrane are able to exclude the dye while cells without an intact membrane take up the coloring agent. </li></ul><ul><li>The dye used for exclusion stain is usually trypan blue but erythrosin and naphthalene black have also been used. </li></ul><ul><li>A dye uptake stain can be used to measure viability as well. In this case, the dye is normally taken up by viable cells but not by the non-viable cells. Diacetyl fluorescein is an example of a dye used for dye uptake assays. </li></ul>
  19. 19. Trypan blue  A stain which will only enter across the membranes of dead/non-viable cells. - Cause cancer in lab. animals - Appropriate precaution should be taken when handling trypan blue (use of extraction hood and gloves)  Dilution by trypan blue - Viable cells : small, round and refractive - Non-viable cells : swollen, larger, dark blue
  20. 20. <ul><li>The most common routine method for cell counting which is efficient and accurate is with the use of a hemocytometer. </li></ul> Hemocytometer Hemocytometer Cell Counts
  21. 21. Volume : 0.1mm 3 1 ml = 1 cm 3 = 1000 mm 3 1 mm 1 mm 0.1 mm
  22. 22. Dead cell
  23. 23. Materials and Equipment <ul><li> Trypan blue (0.4 g trypan blue in 100 ml physiological saline) -> pass through a 0.22 ㎛ filter </li></ul><ul><li> Hemocytometer with coverslip </li></ul><ul><li> Hand-held counter </li></ul><ul><li> Microscope </li></ul>
  24. 24. Methods <ul><li>Clean hemocytometer & coverslip and wipe with 70% alcohol before use </li></ul><ul><li>Place coverslip on hemocytometer </li></ul><ul><li>Mix the cell suspension gently </li></ul><ul><li>Aliquot 0.1 ml cell suspensions </li></ul><ul><li>Add 0.1 ml (2-fold dilution), 0.3 ml (4-fold dilution) or 0.9 ml (10-fold dilution) trypan blue : appropriate range of cells to be counted </li></ul><ul><li>Draw a sample into a Pasteur pipette after mixing </li></ul><ul><li>Draw the cell suspension in to fill the chamber </li></ul><ul><li>Using a light microscope at low power, count the number of cells </li></ul><ul><li>Count the viable & non-viable cells in both halves of the chamber </li></ul>
  25. 25. <ul><li>Calculations </li></ul><ul><li>A = Vol. Of cell solution (ml) </li></ul><ul><li>B = Dilution factor in trypan blue </li></ul><ul><li>C = Mean number of unstained cells </li></ul><ul><li>D = Mean number of dead/stained cells </li></ul><ul><li>10 4 = Conversion of 0.1 mm 3 to ml </li></ul><ul><li>(1) Total number of viable cells </li></ul><ul><li>A Ⅹ B Ⅹ C Ⅹ 10 4 </li></ul><ul><li>(2) Total dead cell count </li></ul><ul><li>A Ⅹ B Ⅹ D Ⅹ 10 4 </li></ul><ul><li>(3) To give a total cell count </li></ul><ul><li>Viable cell count + dead cell count </li></ul><ul><li>(4) % viability </li></ul><ul><li>(Viable cell count/Total cell count) Ⅹ 100 </li></ul>
  26. 26. Example <ul><li>Vol. : Volume </li></ul><ul><li>CS : Cell Solution </li></ul><ul><li>TB : Trypan blue </li></ul>Dilution factor Vol. of CS Cell count Total viable cells 0.1 ml CS + 0.1 ml TB ( 2 ) 20 ml 23 20 Ⅹ 2 Ⅹ23Ⅹ10 4 = 9.2 Ⅹ10 6 cells 0.1 ml CS + 0.3 ml TB ( 4 ) 15 ml // 15 Ⅹ 4 Ⅹ23Ⅹ10 4 = 1.38 Ⅹ10 7 cells 0.1 ml CS + 0.9 ml TB ( 10 ) 10 ml // 10 Ⅹ 10 Ⅹ23Ⅹ10 4 = 2.3 Ⅹ10 7 cells
  27. 27. Automated trypan blue method for optimal cell viability determination www.innovatis.com
  28. 28. Introduction <ul><li> The trypan blue dye exclusion assay is the most commonly used and accepted method for the measurement of cell viability </li></ul><ul><li> It relies on the alteration in membrane integrity as determined by the uptake of dye by dead cells, thereby giving a direct measure of cell viability </li></ul><ul><li> Based on optimal image analysis, the technology allow precise cell-viability and cell-density determination </li></ul><ul><li> The system performs automatic and reproducible measurements of human or animal suspension cell densities as well as standardized differentiation between viable and dead cells, based on the trypan blue dye exclusion method </li></ul><ul><li> The direct and automated optical analysis by means of modern pattern recognition methods allows cell identification and a standardized differentiation between viable and dead cells and also cell debris </li></ul>
  29. 29. <ul><li> The system consist of three functional part: the liquid handling unit, image capture hard ware, and a data processing system, including the user interface </li></ul>Fig. Cedex workbench The IP Result viewer enables the user to control whether the Cedex system recognizes the cells correctly, and whether it reliably differentiates and between viable and dead cells
  30. 30. Result view of the image processing Marked viable and dead cells Viable cell Dead cell
  31. 31. Fig. a) Distribution histogram of the viable cell diameter for a human leukemia cell sample. b) Histogram of compactness for a human leukemia cell sample . The abscissa represents the ratio of cell circumstance to cell area normalized to the value of 1 for an ideal sphere (a) (b)
  32. 32. Vi-CELL TM CELL Viability Analyzer
  33. 34. Principle Trypan Blue dye Exclusion Methods
  34. 35. Run results
  35. 36. <ul><li>One parameter of cell death is the integrity of the cell membrane. </li></ul><ul><li>It can be measured by cytoplasmic enzyme activity released by damaged cells. </li></ul><ul><li>Lactate dehydrogenase is a stable cytoplasmic enzyme present in all the cells. </li></ul><ul><li>The LDH is rapidly released to the culture supernatant upon damage of the plasma membrane. </li></ul>LDH Release
  36. 37. <ul><li> Quantitative value for the loss of cell viability </li></ul><ul><li> The activity of LDH can be measured as the reduction of pyruvate to lactate. </li></ul><ul><li> The reduction is coupled to the oxidation of NADH to NAD + , which is followed spectrophotometrically at 340nm </li></ul><ul><li>LDH </li></ul><ul><li>Pyruvate + NADH + H + ⇌ NAD + + lactate </li></ul><ul><li> As NADH has a high absorbance at 340nm compared to NAD + , the reaction is measured as the rate of decrease in absorbance at 340nm. </li></ul>LDH (lactate dehydrogenase) Leakage
  37. 38. Requirement of LDH assay <ul><li>Greater process productivity (e.g. High-cell-density entrapped-culture systems) : difficulty of cell isolation </li></ul><ul><li>-> Metabolic parameters(glucose uptake) : compromised because uptake/production rates can alter as a result of the cell switching carbon source. </li></ul><ul><li>-> Analysis of the release of intracellular enzymes can be used enzyme in cell culture studies is LDH. </li></ul><ul><li> Assumptions of LDH assay </li></ul><ul><li>: Intracellular enzymes are only released after damage to the cell membrane </li></ul><ul><li>: Rapidly released from damaged cells. </li></ul>
  38. 39. Pitfalls of LDH assay <ul><li> The release of LDH activity can be related to the total No. of dead & lysed cells. </li></ul><ul><li> The stability of LDH can vary considerably, ranging from the loss of a few percent per day to a half-life of 12h depending upon the cell type. </li></ul><ul><li> Assumed that the release of LDH occurs rapidly after damage to the cell membrane. This assumption is not necessarily correct. </li></ul><ul><li> The release of LDH can be complete in cells which are considered dead by dye exclusion methods. </li></ul><ul><li> Complete release may only occur upon cell lysis . </li></ul><ul><li> This point is further complicated because dye exclusion methods do not measure lysed cells. </li></ul>
  39. 40. Reagents and Solutions <ul><li>Buffer (Tris 81.3 mmol/L ; NaCl 203.3 mmol/L ; pH 7.2) </li></ul><ul><li>: Dissolve 4.92 g Tris and 5.95 g NaCl in 400 ml water and adjust to pH 7.2 at 30℃ with HCl. Make up to a final volume of 500 ml with water </li></ul><ul><li>NADH solution (  -NADH 0.17 mg/ml) </li></ul><ul><li>: Dissolve 3.4 mg NADH in 20 ml buffer </li></ul><ul><li>Pyruvate solution (9.76 mmol/L) </li></ul><ul><li>: Dissolve 0.107 g monosodium pyruvate in 90 ml buffer. Make up to a final volume of 100 ml with buffer. </li></ul>
  40. 41. <ul><li> Buffer is stable at 0-4℃. </li></ul><ul><li> The NADH solution is kept at 0-4℃ and must be prepared fresh daily </li></ul><ul><li> The pyruvate solution should be dispensed into 1.5ml aliquots and stored at -20℃. After thawing, each aliquot should be discarded. </li></ul><ul><li> The pyruvate solution is stable for 2 months . </li></ul>Stability of solutions
  41. 42. Materials and Equipment <ul><li> NADH solution </li></ul><ul><li> Pyruvate solution </li></ul><ul><li> Narrow-bandwidth spectrophotometer, fitted with a thermostatted cuvette holder capable of temperature control within  0.1℃ and a chart-recorder. </li></ul>Assay Conditions  Incubation temperature : 30.0℃  Wavelength : 340 nm  Final reaction volume : 1.07 ml  Light path : 1.0 cm
  42. 44. <ul><li>The LDH activity is determined by an enzymatic test. </li></ul><ul><li>The first step is the reduction of NAD + to NADH/H + by the LDH catalysed conversion of lactate to pyruvate. </li></ul><ul><li>In the second step, the catalyst diaphorose trasfers H/H + from NADH/H + to the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phnyltetrazolium chloride (INT), which is reduced to a red formazan. </li></ul><ul><li>The resulting formazan absorbs maximally at 492nm and can be measured quantitatively at 490nm </li></ul><ul><li>References   </li></ul><ul><li>Decker, T. and M. L. Lohmann-Matthes (1988). &quot;A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity.&quot; J Immunol Methods 115 (1): 61-9. </li></ul><ul><li>Korzeniewski, C. and D. M. Callewaert (1983). &quot;An enzyme-release assay for natural cytotoxicity.&quot; J Immunol Methods 64 (3): 313-20. </li></ul><ul><li>Lappalainen, K., I. Jaaskelainen, et al. (1994). &quot;Comparison of cell proliferation and toxicity assays using two cationic liposomes.&quot; Pharm Res 11 (8): 1127-31. </li></ul><ul><li>Nachlas, M. M., S. I. Margulies, et al. (1960). &quot;The determination of lactic dehydrogenase with a tetrazolium salt.&quot; Anal Biochem 1 : 317-26. </li></ul>
  43. 45. <ul><li> Ethidium bromide (EtBr) and propidium iodide (PI) </li></ul><ul><li>PI is impermeable to intact plasma membrane . </li></ul><ul><li> PI binds to nucleic acids upon membrane damage : flow cytometric techniques depend on fluorescence, PI is ideally suitable for the rapid evaluation of the permeability properties of large numbers of cells while maintaining good statistical accuracy. </li></ul><ul><li>Intercalates with DNA or RNA  red </li></ul>Fluorescent dyes
  44. 46. <ul><li> Fluorescein diacetate (FDA) is a nonpolar ester which passes through plasma membranes and is hydrolyzed by intracellular esterases to produce free fluorescein, the polar fluorescein is confined within cells which have an intact plasma membrane and can be observed under appropriate excitation conditions. </li></ul><ul><li>Undamaged cell : highly fluorescent fluorescein dye </li></ul><ul><li>Damaged cell : fluoresce only weakly </li></ul><ul><li> greenish-yellow at 450-480 nm </li></ul>
  45. 47. Schematic illustration of the principle of PI/FDA cell viability assay Intact cell – PI and FDA is added Fluorescein in intact cells ● FDA (Fluorescein diacetate ) ● PI (Propidium iodide) Plasma membrane is damaged ; fluorescein leaks out PI enters and strains nucleic acids
  46. 48. Example 1 ; Observation of cell death A group of hepatoma cells exposed to a diffusing wave of digitonin. Intact cells (green) are damaged by digitonin, loose the green fluorescence and acquire red fluorescence of PI.
  47. 49. Evaluate viability by examining the metabolic components that are necessary for cell growth , on the premise that cellular damage will inevitably result in the loss of ability to maintain and provide energy for metabolic function and growth. Functional assays
  48. 50. Colorimetric assay <ul><li> Rapid and accurate assessment of viable cell number </li></ul><ul><li> Miniaturized into 96-well plates </li></ul><ul><li> Measure using an Microplate reader </li></ul><ul><li> Permit many sample to be analyzed rapidly </li></ul><ul><li> Reduce medium and plastics costs </li></ul>
  49. 51. <ul><li> These assay are read at 570 nm (except for the Acid Phosphatase (A.P) assay-wavelength is 405 nm) on a ELISA plate reader, using a 620 nm filter as reference wavelength. </li></ul><ul><li> It is important to remove any bubbles from the well before absorbance readings. </li></ul><ul><li> Examples are </li></ul><ul><li> MTT / XTT assay </li></ul><ul><li>Crystal violet dye elution (CVDE) </li></ul><ul><li>Acid phosphatase (AP) assay </li></ul><ul><li>Alamar blue oxidation-reduction assay </li></ul><ul><li>Neutral Red (NR) assay </li></ul><ul><li>[ 3 H]-thymidine and BrdU incorporation </li></ul>
  50. 52. <ul><li>Introduction </li></ul><ul><li> This assay is a sensitive, quantitative and reliable colorimetric assay that measures viability, proliferation and activation of cells. </li></ul><ul><li> The assay is based on the capacity of mitochondrial dehydrogenase enzymes in living cells to convert the yellow water-soluble substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan product which is insoluble in water. </li></ul>MTT Assay
  51. 53. <ul><li> The amount of formazan produced is directly proportional to the cell number in range of cell lines. </li></ul>metabolically active Cell MTT F ormazan Insoluble
  52. 54. <ul><li>Materials and equipment </li></ul><ul><li>MTT solution (5 ㎎ / ㎖ in phosphate buffered saline (PBS) pH 7.5), HCl, Propan-2-ol </li></ul><ul><li>96-well microtiter plate, ELISA reader </li></ul><ul><li>Procedure (suspension and monolayer cells) </li></ul><ul><li>1. Prepare MTT stock solution and fiter through a 0.2 ㎛ filter to sterilize and remove the small amount of insoluble residue </li></ul><ul><li>2. To 100 ㎕ cell suspension or cell monolayer in each microtiter well add 10 ㎕ MTT </li></ul><ul><li>3. Incubate in a humidified incubator at 37℃ for 3 h </li></ul><ul><li>4. Add 100 ㎕ 0.04 M HCl in propan-2-ol to each well and mix thoroughly to dissolve insoluble dark blue formazan crystals </li></ul><ul><li>5. Read plate on a ELISA reader using a test wavelength of 570 nm and reference wavelength of 630 nm </li></ul>
  53. 55. <ul><li>Advantages of MTT assay </li></ul><ul><li>Considered a major advance; used the most prevalent in vitro assay </li></ul><ul><li>Rapid, versatile, quantitative and highly reproducible </li></ul><ul><li>Adaptable to large-scale screening; relevant for most cells </li></ul><ul><li>MTT reduction correlates to indices of cellular protein and earlier cell number </li></ul><ul><li>More sensitive and earlier predictor of toxicity than classical LDH or neutral red measurements </li></ul>
  54. 56. <ul><li>Disadvantage of MTT assay </li></ul><ul><li>Production of the MTT product is dependent on the MTT concentration in the medium. The kinetics and degree of saturation are dependent on cell type. </li></ul><ul><li>Assay is less effective in the absence of cell proliferation. </li></ul><ul><li>MTT cannot distingulish between cytostatic and cytocidal effect. </li></ul><ul><li>Individual cell numbers are not quantitated and results are expressed as a percentage of control absorbance. </li></ul><ul><li>Test is less effective if cells have been cultured in the same media that has supported growth for a few day, which leads to underestimation of control and untreated samples. </li></ul>
  55. 57. Example; NIH J2 3T3 cell in medium MTT solution elution Absorbance at 540 nm
  56. 58. XTT assay <ul><li>Introduction </li></ul><ul><li> The assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazan dye metabolic active cells. </li></ul><ul><li> This conversion only occurs in viable cells. </li></ul><ul><li> The formazan dye formed is soluble in aqueus solution and is directly quantified using Microplate reader. </li></ul><ul><li> Both MTT and XTT work by being to a formazan dye only by metabolic active cells. </li></ul>
  57. 59. metabolically active cell Water soluble
  58. 60. <ul><li>Materials and equipments </li></ul><ul><li>XTT labeling regent, electron-coupling reagent </li></ul><ul><li>96-well microtiter plate, Microplate reader </li></ul><ul><li>-> XTT labeling mixture : mixed 5 ㎖ XTT labeling reagent with 0.1 ㎖ electron coupling reagent </li></ul><ul><li>Procedure </li></ul><ul><li>Cell are grown in microtiter plates in a final volume of 100 ㎕ culture medium per well. The incubation period of the cell cultures depends on the particular experimental approach and on the cell line. </li></ul><ul><li>After incubation period, add to each well 50 ㎕ of the XTT labeling mixture </li></ul><ul><li>Incubate the microtiter plate for 4 to 24 h in incubator </li></ul><ul><li>Read plate on a Microplate reader using a wavelength between 450 and 500 nm (reference wavelength of 650 nm) </li></ul>
  59. 61. <ul><li>Compare with MTT assay and XTT assay </li></ul>Culture cells in a MTP for a certain period of time (37℃) MTT assay XTT assay Prepare labeling mixture Incubate cells (0.5-4 h, 37℃) Add solubilizing solution (Isopropanol) and incubate Measure absorbance using an ELISA reader Add XTT labeling mixture Add MTT labeling reagent Insoluble formazan Soluble formazan
  60. 62. Example: MTT and XTT MTT XTT Jenny G., Mark H., Anna J., Inger K., Douglas Mc., Roland M., 2002. Evaluation of redox indicators and the use of digital scanners and spectrophotormeter for quantification of microbial growth in microplates. J. Micro. Methods. 50:63-73
  61. 63. Principle <ul><li> Dye elution: </li></ul><ul><li>Cell up-taken dye was measured colorimetric method after acetic acid dye elution. </li></ul><ul><li> Nuclei counting </li></ul><ul><li>Incubation of cell samples in a mixture of citric acid and crystal violet causes cells to lyse and the released nuclei to stain purple. </li></ul>Crystal violet
  62. 64. Procedure <ul><li> Dye elution </li></ul><ul><li>① After removal of medium, rinse 96 well plates with 100 ㎕ /well of PBS and stain with 100 ㎕ 0.25% (g/10ml) aqueous crystal violet for 10 min. </li></ul><ul><li>② Rinse plats four times in tap water. </li></ul><ul><li>③ Dry the outsides of the plates with paper to help avoid water stains, and then dry the plates at 37℃. When dry, add 100 ㎕ per well of 33% glacial acetic acid (33 ml/100ml) and mix the contents of each well before reading at 570 nm. </li></ul><ul><li> Nuclei counting </li></ul><ul><li>① Allow microcarriers from a culture sample (1ml) to settle to the bottom of a centrifuge tube. </li></ul><ul><li>② Removed clear supernatant by aspiration. </li></ul><ul><li>③ Add 1ml of crystal violet reagent. </li></ul><ul><li>④ Incubate at 37℃ at least 1 h. </li></ul><ul><li>⑤ Introduce a sample into the </li></ul><ul><li>hemocytometer chamber and count the purple-stained nuclei as for whole cells. </li></ul>
  63. 65. Example; Monolayer culture Ref.:Journal of Orthopaedic Resarch19 (2001) ※ OP : Osteoporosis Crystal Violet - 60minutes Non-OP OP
  64. 66. Example; Microcarrier culture Rabbit oral mucosal cell cultured by microcarrier Cell counting
  65. 67. Acid phosphatase (AP) assay <ul><li>Introduction </li></ul><ul><li> The action of this enzyme in many of tissue is to cleave a waste product called pyrophosphate and effectively convert it to a useable phosphate. </li></ul><ul><li> P-nitrophenyl phosphate will be the substrate and nitrophenol is the product of this reaction. </li></ul><ul><li> Nitrophenol is colorless but when the pH of the reaction solution is alkaline, it is appears yellow. The pH of the reaction solution will be changed by the addition of NaOH. </li></ul>
  66. 68. P-nitrophenyl phosphate + Acid phosphatase  Nitrophenol + HPO 4 -2
  67. 69. <ul><li>Materials and equipment </li></ul><ul><li>Substrate-containing buffer : 10 mM P-nitrophenyl phosphate in 0.1 M sodium acetate pH 5.5, 1 M NaOH </li></ul><ul><li>96-well micro titer plate, Microplate reader </li></ul><ul><li>Procedure </li></ul><ul><li>1. At end of cell growth period, remove medium and rinse wells in 100 ㎕ PBS </li></ul><ul><li>2. Add 100 ㎕ substrate-containing buffer to each well </li></ul><ul><li>3. Incubate for 2 h in incubator. Read plates at 405 nm, and either reincubate for a further time if increased sensitivity is required, or ‘stop’ with addition of 50 ㎕ /well of 1 M NaOH to cause an electrophilic shift in the p-nitrophenol chromophore and thus develop the yellow color, giving greatly increased sensitivity </li></ul>
  68. 70. <ul><li> Principle </li></ul><ul><li>In the presence of cellular metabolism the color of Alamar Blue (ALB) changes from a fully oxidized, non fluorescent blue to a fully reduced, fluorescent red . ALB will be reduced by a variety of enzymes and small molecules, including the cytochrome system, FMN, FAD, NAD, and NADP. </li></ul><ul><li> Advantages </li></ul><ul><li>Simple, rapid, inexpensive, required no lysis, extraction or washing of sample </li></ul><ul><li> Disadvantages </li></ul><ul><li>Unstable during storage (absorbance of oxidized ALB-3day, reduced ALB-increase from one day to the next) </li></ul><ul><li> Characteristics </li></ul><ul><li>- Sensitivity : </li></ul><ul><li>Propidium iodide (PI), Sulforhodamine B (SRB) > ALB=MTT </li></ul><ul><li>- The ALB assay is faster, simpler, and less artefact prone than the MTT assay. </li></ul>Alamar Blue oxidation-reduction assay
  69. 71. Procedure
  70. 73. Neutral Red assay ( 3-amino-7dimethyl-2-methyphenazine hydrochloride) <ul><li> Principle </li></ul><ul><li>- The incorporaton of NR into the lysosomes of viable cells after their incubation with test agents . </li></ul><ul><li> Use </li></ul><ul><li>- Industrial, pharmaceutical, environmental and other testing laboratories concerned with acute toxicity testing. </li></ul><ul><li> Advantages </li></ul><ul><li>- Simplicity, speed, economy, and sensitivity </li></ul>
  71. 74. Materials and Equipments <ul><li> Solution </li></ul><ul><li>① Neutral red </li></ul><ul><li>4mg/ml stock solution </li></ul><ul><li>Dilute 1:100 into medium , incubate overnight at 37℃ and centrifuge for 10 min at 1500 g before use. </li></ul><ul><li>② 1% CaCl 2 /0.5% formaldehyde </li></ul><ul><li>Mix 6.5 ml 37% formaldehyde with 50 ml 10% CaCl 2 and 445 ml distilled water. </li></ul><ul><li>③ 1% acetic aicd/50% ethanol </li></ul><ul><li>Mix 4.75 ml acetic acid with 250 ml 95% ethanol and 245 ml distilled water. </li></ul><ul><li> Equipment </li></ul><ul><li>① Complete media suitable for chosen cell type. </li></ul><ul><li>② Culture petri dish </li></ul><ul><li>③ 96well tissue culture plate </li></ul><ul><li>④ Inverted microscope </li></ul><ul><li>⑤ ELISA-type spectrophotometer </li></ul><ul><li>⑥ Microplate shaker </li></ul><ul><li>⑦ Eight-channel pipette </li></ul>
  72. 75. Procedure <ul><li>① Resuspend cells of actively growing culture and count cells and accurately allocate appropriate number suspended in medium. </li></ul><ul><li>② Seed 0.2 ml containing desired number of cells to each well of 96 well plate and incubate at 37℃ for 24 h or longer. </li></ul><ul><li>③ Removed the medium and add fresh medium containing graded dilutions of test agent. Incubate for desired length of time. Examine at least 4-8 wells per concentration of test agent. </li></ul><ul><li>Keep serum concentration as low as possible during this step. </li></ul><ul><li>(prevent or to reduce adsorption of xenobiotic to serum components) </li></ul><ul><li>④ After incubation for desired time interval, remove medium with test agent and incubate cells with fresh medium containing 40 ㎍ /ml NR dye. </li></ul><ul><li>⑤ Continue incubation for 3h to allow for incorporation of vital dye into survival cells. </li></ul><ul><li>⑥ Remove medium by inverting the plate and rapid rinse with a mixture of 1% CaCl 2 / 0.5%formaldehyde. </li></ul><ul><li>⑦ Extract dye into suprernate with 0.2 ml of solution of 1% acetic acid/50% ethanol. </li></ul><ul><li>After10 min at room temperature and rapid agitation for a few seconds on a micrometer plate shaker, scan the plate with an ELISA-type spectrophotometer equipped 540 nm filter. </li></ul>
  73. 76. Principle <ul><ul><li>The rate of DNA synthesis is a reflection of proliferation under many condition.To measure the proliferative rates by [ 3 H]-thymidine uptake, cells are cultured in microtitre wells, thymidine is added, and the uptake by DNA is measured , after lysing and washing on, by scintillation counting. Bromodeoxyuridine(BrdU) can be incorporated instead of [ 3 H]-thymidine and the incorporation can be assayed with antibodies to BrdU in a non-radioactive assay. </li></ul></ul>[ 3 H]-thymidine and BrdU incorporation (DNA synthesis measurement)
  74. 77. Schematic diagram of [ 3 H]-TdR and BrdU
  75. 78. Labeling index with [ 3 H]-thymidine <ul><li>① Set up the culture at 2x10 4 cells/ml~ 5x10 4 cells/ml in 24 well plates containing cover-slips. Grow to the desired cell density. </li></ul><ul><li>② Add [ 3 H]-thymidine to the medium . 100KBq/ml(~5μCi/ml)and incubate for the cultures 30 min. </li></ul><ul><li>③ Remove the labeled medium, and discard it into a designed container for radioactive waste. </li></ul><ul><li>④ Wash the cover-slips three times with PBSA. </li></ul><ul><li>⑤ Add 1:1 PBSA: acetic methanol, 1ml per well, and remove it immediately </li></ul><ul><li>⑥ Add 1ml of acetic methanol at 4 to each well, and leave the cultures for 10min. </li></ul><ul><li>⑦ remove the cover-slips, and dry them with a fan </li></ul><ul><li>⑧ mount the cover-slip on a microscope slide with the cells uppermost. </li></ul><ul><li>⑨ Leave the mountant to dry overnight. </li></ul>
  76. 79. DNA synthesis by [ 3 H]-thymidine <ul><li>① Grow the culture to the desired density. </li></ul><ul><li>② [ 3 H]-TdR, 40 KBq /ml(~1.0μCi), 2 MBq/mol(~50 μCi/mol) in HBSS. </li></ul><ul><li>※ 1Ci =3.7*10 10 회 / s = 3.7*10 10 Bq , 1Bq =1 회 /s </li></ul><ul><li>③ Incubate the cell for 1-24 h. </li></ul><ul><li>④ Remove the radioactive medium carefully. </li></ul><ul><li>⑤ Wash the cell carefully with 2 ml of HBSS, PBSA, and 2 ml ice-cold 0.6 M TCA for 10 min. </li></ul><ul><li>⑥ Wash the cell with TCA twice 5 min each time. </li></ul><ul><li>⑦ 0.5 ml of 2 M perchloric acid, a hot plate at 60℃ for 30min and allow the solution to cool. </li></ul><ul><li>⑧ Add 0.5ml SLS in NaOH incubate the solution at 37℃ for 30 min or overnight at room temperature. </li></ul><ul><li>⑨ Collect the solubilized pellet and determine the radioactivity. </li></ul>
  77. 80. DNA labeling assay (using fluorescent probes assay)
  78. 81. Two types of nonviable cells
  79. 82. Two methods: Enzymatic DNA labeling and DNA-binding dye labeling
  80. 83. Ⅰ . Enzymatic DNA labeling dNTP dUTP Direct Indirect X Fluoresein, FITC, PE etc Biotin DIG Avidin conjugated with fluoresein, AP, POD Anti-DIG antibody conjugated with fluoresein, AP, POD
  81. 84. Fig. Immunostaining of apoptotic cells (dark brown) by TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) and peroxidase staining in rabbit endometrium
  82. 85. DNA-binding dyes: fluorochrome 1 RNase treatment is required 2 RNase treatment is required because PI could stain double strand RNA Ⅱ . DNA-binding dye labeling Dye Permeability via intact membrane Staining DNA RNA Acridine orange Yes Green Red-orange 1 Hoechst 33342 Yes Blue No Hoechst 33258 Yes Blue No DAPI (4,6-diamidino-2-phenylindole) Yes Bright blue No EtBr (Ethidium bromide) No Orange Slightly red 1 PI (Propidium iodide) No Red No 2
  83. 86. Pattern of dye staining according to color and chromatin morphology * late apoptosis is regarded as the stage of membrane fragmentation and secondary necrosis Dye Apoptosis Necrosis Early apoptosis Late apoptosis* Acridine orange Green Condensed Green Fragmented Green Diffuse, Intact Hoechst 33342 Blue Condensed Blue Fragmented Blue Diffuse, Intact Hoechst 33258 Blue Condensed Blue Fragmented Blue Diffuse, Intact DAPI Blue Condensed Blue Fragmented Blue Diffuse, Intact Ethidium bromide No (Orange, Condensed if permeabilized) Orange Fragmented Orange Diffuse, Intact Propidium iodide No (Red, Condensed if permeabilized) Red Fragmented Red Diffuse, Intact
  84. 87. Fig. In Hoechst 33258 / PI double staining, cells with blue intact nuclei were viable cells, whereas those with blue fragmented nuclei were early apoptotic cells. Cells with pink intact nuclei were necrotic cells, whereas cells with pink fragmented nuclei were late apoptotic cells. (blue against Hoechst33258, red against PI) Apoptotic(0%) Necrotic(10.5%) Apoptotic(85.2%) Necrotic(11.2%) Apoptotic(1.2%) Necrotic(92.5%)
  85. 88. Fig. DAPI staining of condensed nuclei of apoptotic cells
  86. 89. Acridine orange
  87. 90. <ul><li> Fluorescent protein biosensors measuring the molecular dynamics of macromolecules, metabolites, and ions in single cells have emerged from the integrative use of contemporary synthetic organic chemistry, biochemistry, and molecular biology. </li></ul><ul><li> Vascular endothelial cells (ECs) play and important role in physiologic hemostasis and blood vessel permeability, express immune related functions in monocytes and macrophages, and the viability of ECs is important in predicting the post-operative function and durability of cryopreserved vessels for implantation. </li></ul><ul><li> Tetrameric Griffonia simplicifolia agglutins (GS1) shows prominent binding only to the a- D-galactosyl residue of blood vessel ECs. </li></ul><ul><li> Staining with fluorescein isothiocyanate(FITC) conjugated with GS1 differentiates ECs from the other cells in flow cytometry. </li></ul>
  88. 91. <ul><li> PI intercalates DNA double strands in dead cells without regard to cell types, as their membrane lose integrity. </li></ul><ul><li> GS1-FITC and PI double staining : presumed to immediately determine the differential viability of ECs from whole cells </li></ul><ul><li> The use of fluorescent probes enables the rapid determination of the viability of ECs and whole cells from the same tissue without separating ECs from whole cells, and of the viability of each step in the cryopreservation process </li></ul>
  89. 92. Fig. Each part of quadrant statistics was observed under fluorescence microscopy. Live ECs preferentially expressed the green color of GS1 . Dead ECs are double stained by the green color of GS1-FITC and the red color of PI, which results in yellow . Dead cells except dead ECs are identified by only the red color of PI.
  90. 93. Morphological assay
  91. 94. <ul><li> Large-scale, morphological changes that occur at the cell surface, or in the cytoskeleton , can be followed and related to cell viability. </li></ul><ul><li> Damage can be identified by large decreases in volume secondary to losses in protein and intracellular ions of due to altered permeability to sodium or potassium. </li></ul><ul><li> Necrotic cells : nuclear swelling, chromatin flocculation, loss of nuclear basophilia </li></ul><ul><li> Apoptotic cells : cell shrinkage, nuclear condansation, nuclear fragmentation </li></ul>
  92. 95. Example; Morphological feature (Human skin keratinocyte) Fig. Morphological feature of (A) normal human skin keratinocyte, and differentiated human skin keratinocyte(B) . (A) (B)
  93. 96. Example; Morphological feature (Human skin fibroblasts) Fig. Morphological feature of (A) normal human skin fibroblasts, and aging human skin fibroblasts(B) . (A) (B)
  94. 97. Reproductive Assay
  95. 98. <ul><li>Clonogenic Cell: </li></ul><ul><li> Defined as a cell with the capacity for sustained proliferation </li></ul><ul><li> Have undergone a minimum of 5-6 doublings to give rise to colonies containing at least 50 cells </li></ul>Colony-forming Efficiency
  96. 99. <ul><li>Colony-forming Efficiency (CFE) </li></ul><ul><li> The ability to form colonies is used as a measure of </li></ul><ul><li>reproductive integrity </li></ul><ul><li> It is often referred to as plating efficiency (PE) </li></ul><ul><li>Number of colonies formed </li></ul><ul><li>CFE =  100% </li></ul><ul><li>Number of cell plates </li></ul><ul><li>Clonogenic assays </li></ul><ul><li> Be used to reflect stem cell content </li></ul><ul><li> The basis of assays for determining the lethal effects of </li></ul><ul><li>cytotoxic agents </li></ul>
  97. 100. Determining the PE of an established adherent cell line <ul><li>Materials and Equipment </li></ul><ul><li> Cell growth medium : Eagle’s basal medium (BME) </li></ul><ul><li>100 iu/ml penicillin, </li></ul><ul><li>0.1 mg/ml streptomycin </li></ul><ul><li> Trypsin-EDTA </li></ul><ul><li> Gentain violet stain </li></ul>
  98. 101. Procedure <ul><li>1. Trypsinize monolayer cultures or use cell suspension </li></ul><ul><li>cultures and determine the viable cell count </li></ul><ul><li>2. Dilute cells in growth medium to 1000 , 2000 and </li></ul><ul><li>5000 cells/10ml </li></ul><ul><li>3. Inoculate nine replicate Petri dishes with 4 ml growth medium plus 1ml cell suspension </li></ul><ul><li>4. Place plates in a humidified 5% CO 2 plus air incubator are normal growth temperature and rock shelf or tray gently to and fro three times. The plates must not be moved now until colonies are stained </li></ul><ul><li>5. Stain and count three replicate per cell density at 1,2 and 3 weeks (murine lines) or 2 , 3 and 4 weeks (human lined) </li></ul><ul><li>6. Calculate the optimum cell densities for seeding and duration of incubation </li></ul>
  99. 102. Example; Rat keratinocytes (A) (B) (C) (D) : colony , : Single cells Colony forming Non-colony forming 48 hr after subculture 6 days after subculture
  100. 103. Reference <ul><li>1. Cell quantification, module 4B:1, Hemocytometer cell counts and viability studies, 1.1 – 1.5 </li></ul><ul><li>2. http://www.embl-heidelberg.de/ ExternalInfo/karsenti/countingcells. html </li></ul><ul><li>3. http://www.bd.com/clinical/POL/products/equipment/hemacytm.asp </li></ul><ul><li>4. Park JC, Hwang YS, and Suh H., Viability Evaluation of engineered </li></ul><ul><li>Tissue., Yonsei Medical Journal 2000 41(6): 836-844 </li></ul><ul><li>5. Ashish A., Sonia SY, Mark AH, and Minas TC., Pressure Related </li></ul><ul><li>apoptosis in Neuronal Cel Lines., Journal of Neuroscience Research </li></ul><ul><li>2000 60: 495-503 </li></ul><ul><li>6. Islam TC, Skarin T, Sumitran S, and Toftgard R., Retinoids induce </li></ul><ul><li>apoptosis in cultured keratinocytes., British Journal of Dermatology </li></ul><ul><li>2000 143:1709-719 </li></ul>
  101. 104. <ul><li>7. Maria L. Anthony, Shane N. O. Williams, and Kevin M. Brindle, Nuclear magnetic resonance methods of monitoring cell metabilism., Methods in Biotechnology, Vol. 8: Animal Cell Biotechnology, 165-175, Edited by: N. </li></ul><ul><li>Jenkins, Humana Press Inc. Totowa, NJ. 1999. </li></ul><ul><li>8. Macdonald JM, Grillo M, Schmidlin O, Tajiri DT, James TL., NMR spectroscopy and MRI investigation of a potential bioartificial liver., NMR Biomed. 1998 Apr; 11(2):55-66. </li></ul><ul><li>9. Flendrig LM, la Soe JW, Jorning GG, Steenbeek A, Karlsen OT, Bovee WM, Ladiges NC, te Velde AA, Chamuleau RA., In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates., J Hepatol . 1997 Jun;26(6):1379-92. </li></ul><ul><li>10. Fluorescence and confocal microscopy </li></ul><ul><li>( http://helios.mol.uj.edu.pl/conf_c/main.htm ) </li></ul><ul><li>11. Breuls RG, Mol A, Petterson R, Oomens CW, Baaijens FP, Bouten CV., Monitoring local cell viability in engineered tissues: a fast, quantitative, and nondestructive approach., Tissue Eng . 2003 Apr;9(2):269-81. </li></ul><ul><li>12. Griffiths JB, Newell DG, and Doyle A., Cell & Tissue Culture: Laboratory procedures., Cell Quantification, 4B, JOHN WILEY & SONS Ltd, UK.1993 </li></ul>
  102. 105. In-vivo models
  103. 106. Models of transplantable tumour <ul><li>Murine solid tumour models with Daltons lymphomatic cells </li></ul>
  104. 107. Ascites tumor model <ul><li>Normal mouse </li></ul><ul><li>Tumor bearing mouse </li></ul>

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