Standard Lymphocyte Culture


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Making Chromosome Slides

Standard Lymphocyte Culture

  1. 1. Introduction <ul><li>Peripheral blood: </li></ul><ul><li>Blood sample are comparatively easy to obtain </li></ul><ul><li>Inexpensive culture techniques --> generate abundant </li></ul><ul><li> metaphases </li></ul>------ high resolution G-banding and FISH studies <ul><li>Peripheral blood: </li></ul><ul><li>Erythrocytes, platelets and leukocytes (white blood cells, nucleated ) </li></ul><ul><li>Neutrophils, eosinophile, basophils, monocytes and lymphocytes </li></ul>----- Under appropriate conditions, active division of lymphocyte can be induced in vitro
  2. 2. Stimulation of lymphocytes and the use of mitogens <ul><li>Lymphocytes: </li></ul><ul><li>T hymus dependent cells (T-cells): </li></ul><ul><li> responsible for cellular immunity, 70% of lymphocytes </li></ul><ul><li>2. B ursa dependent cells (B-cells): </li></ul><ul><li>responsible for antibody production </li></ul>In vivo: Antigens ---> small to enlarge ---> non-dividing to proliferation and division In vitro: Mitogens --->PHA ( a mucoprotein) or PWM transforming large populations of lymphocytes into mitotically active cells.
  3. 3. Standard lymphocyte culture PHA exposure ------> morphological transformation ------> RNA synthesis, nuclei enlarge and DNA synthesis commences ------> wave of mitotic activity 48, 72 and 96 hr 1. Collection, transport and storage of samples Adult or older children: 5-10 ml with heparin Fetal and neonatal samples: 1-2 ml with heparin Samples are stable for several days
  4. 4. Culture medium: constituents and storage 2. Culture medium: constituents and storage <ul><li>Basal medium: RPMI 1640 (low thymidine content) </li></ul><ul><li>Supplements </li></ul><ul><li>Fetal calf serum (5-30% of complete medium) </li></ul><ul><li>Antibotics </li></ul><ul><li>Gram-positive bacteria: penicillin (100U/ml) </li></ul><ul><li>Gram-negative bacteria: streptpmycine (100  g/ml) </li></ul><ul><li>(5) L-Glutamine: essential and unstable, amino acid </li></ul><ul><li>(6) Buffering systems: HEPES and sodium bicarbonate </li></ul><ul><li>(7) Phytohaemagglutinin (PHA) </li></ul>
  5. 5. Initiation of a routine 72-h culture <ul><li>Aliquot 6 ml of complete medium into a universal bottle. </li></ul><ul><li>Inoculate the bottle with an appropriate amount of whole </li></ul><ul><li>blood, and mix well: </li></ul><ul><li>Adult blood, 6 drops </li></ul><ul><li>Fetal or neonatal blood, 4-5 drops Washed EDTA blood, 8 drops </li></ul><ul><li>3. Incubate the culture at 37 o C for 72 h. </li></ul><ul><li>Add 0.1 ml Cocemid (10  g/ml) and incubate at 37 o C for a </li></ul><ul><li>further 30 min. Cocemid : colchicine analogue, deacetylmethylcolchicine </li></ul><ul><li>5. Harvest the sample </li></ul>
  6. 6. Harvesting lymphocyte culture The point of harvest is engineered so that a maximum number of cells are in metaphase. <ul><li>Increase the mitotic index: </li></ul><ul><li>Estimation of when most cells are dividing, 24-h interval </li></ul><ul><li>peak mitotic activity at approximately 72 h </li></ul><ul><li>Addition of a spindle inhibitor, colchicine, vinblastine, </li></ul><ul><li>colcemid. </li></ul><ul><li> Cell is subjected to a hypotonic solution (0.075M KCl) -- </li></ul><ul><li>causing cells to swell and ensuring that chromosome are </li></ul><ul><li>adequately dispersed within the lymphocytes </li></ul><ul><li>First fixation (methanol: acetic acid = 3 : 1), </li></ul><ul><li>second fixation and third fixation </li></ul>
  7. 7. Protocol for harvesting lymphocyte cultures <ul><li>Transfer the culture to a centrifuge tube </li></ul><ul><li>Centrifuge the sample, pour off the majority of </li></ul><ul><li>supernatant (leaving approximately 1 ml) </li></ul><ul><li>Thoroughly resuspend the cell pellet, add approximately </li></ul><ul><li>7 ml 0.075 M KCl and mix well </li></ul><ul><li>Incubate at 37 o C for 5 min </li></ul><ul><li>Centrifuge the sample, pour off the majority of </li></ul><ul><li>supernatant (leaving approximately 1 ml) </li></ul><ul><li>Thoroughly resuspend the cell pellet </li></ul><ul><li>While continue to agitate the cells,slowly add </li></ul><ul><li>approximately 1 ml fixative (drop-wise). Color turn dark </li></ul><ul><li>Centrifuge the sample, pour off the supernatant </li></ul><ul><li>Resuspend the cell pellet with fresh fixative and mix well. </li></ul><ul><li>Preparations should be stored at -20 o C until slide making </li></ul>
  8. 8. Thymidine synchronization of cell division Since all cell do not react to PHA at the same time, this synchrony of cells division is fairly inexact > 1 mM The effect of excess thymidine is to greatly increase the proportion of cells in S-phase
  9. 9. Thymidine synchronized lymphocyte culture <ul><li>Initiate culture </li></ul><ul><li>After incubation at 37 o C for 48 h, add 0.1 mi thymidine solution </li></ul><ul><li>to each culture. Mix well and incubate the culture for a futher </li></ul><ul><li>16 h at 37 o C </li></ul><ul><li>Transfer each culture to centrifuge tube </li></ul><ul><li>Centrifuge the sample and carefully pour off the supernatant </li></ul><ul><li>Resuspend the cell pellet in 7 ml of PBS, warmed to 37 o C </li></ul><ul><li>Centrifuge the sample and carefully pour off the supernatant </li></ul><ul><li>Resuspend the cell pellet in 7 ml of pre-warmed wash medium </li></ul><ul><li>Incubate the culture for 3 h 45 min </li></ul><ul><li>Add 0.1 ml cocemid, and incubate for a further 15 min at 37 o C </li></ul><ul><li>Harvest the culture </li></ul>
  10. 10. FDU ( f luoro d eoxy u ridylate) and MTX ( m etho t re x ate) Foloic acid cycle MTX FDU
  11. 11. Slide making <ul><li>Centrifuge the sample and pour off the supernatant </li></ul><ul><li>Resuspend the pellet in approximately 10 ml fresh fixative </li></ul><ul><li>Centrifuge the sample and pour off the supernatant </li></ul><ul><li>Resuspend the cell pellet and fresh fixative drop by drop until the </li></ul><ul><li>desired cell density for slide making is achieved </li></ul><ul><li>Apply one or two drops of cell suspension to the centre of a cleaned, </li></ul><ul><li>appropriately labelled slide and allow to dry </li></ul><ul><li>6. Repeat step 5 to produce as many as required </li></ul>
  12. 12. Typical examples of karyotype
  13. 13. Factors affecting slide making <ul><li>Slide condition: slide are soaking in a detergent solution, </li></ul><ul><li>than rinse with distill water </li></ul><ul><li>2. Fixative: fresh made fixative, 6:1 - 2:1 </li></ul><ul><li>Temperature and humidity: 20 o C, humidity level: 40-50% </li></ul><ul><li>Cell density </li></ul><ul><li>Slide angle </li></ul><ul><li>Height from which the cell suspension is dropped </li></ul>