Nguyen Thi Nhi completed the following works in the third week of her master's program: 1) Learned techniques for MIN6 cell culture including making media and using an autoclave. 2) Studied cell culture and immunoassay techniques in theory. 3) Specifically learned how to make cell culture media including DMEM, FBS, penicillin/streptomycin, and beta-mercaptoethanol and the purpose of each component. She also learned proper operation of the JSR autoclave for sterilization.

1. NGUYEN THI NHI - Master’s student
Department of Physiology
Keimyung University School of Medicine
Progress report

2. Finished works in 3rd week
1. Practice
1.1. Learned making media for MIN6 cell culture
1.2. Learned how to use JSR autoclave
2. Theory
2.1. Cell culture
2.2. Immunoassay techniques

3. 1.1. Making media
• high glucose DMEM
• 15% FBS (Fetal Bovine Serum)
• 1% P/S (Penicillin/Streptomycin)
• B-mercaptoethanol
352ml/100ml

4. high glucose DMEM
• DMEM contains 4 times the concentration of amino acids and vitamins
than the original Eagle's Minimal Essential Medium.
• DMEM was originally formulated with low glucose (1 g/L) and sodium
pyruvate, but is often used with higher glucose levels, with or without
sodium pyruvate.
• DMEM contains no proteins, lipids, or growth factors. Therefore,
DMEM requires supplementation, commonly with 10% Fetal Bovine
Serum (FBS). DMEM uses a sodium bicarbonate buffer system (3.7 g/L),
and therefore requires a 5–10% CO2 environment to maintain
physiological pH.

5. FBS (Fetal Bovine Serum)
• Fetal bovine serum (FBS) is the liquid fraction of clotted blood from
fetal calves, depleted of cells, fibrin and clotting factors, but
containing a large number of nutritional and macromolecular factors
essential for cell growth.
• Bovine serum albumin is the major component of FBS. Growth
factors in FBS are essential for the maintenance and growth of
cultured cells.
• FBS also contains a variety of small molecules like amino acids, sugars,
lipids, and hormones.

6. beta-mercaptoethanol
• Gibco™ 2-Mercaptoethanol (also known as beta-mercaptoethanol or
BME) is a potent reducing agent used in cell culture media to prevent
toxic levels of oxygen radicals.
• 2-Mercaptoethanol is not stable in solution, so most protocols require
daily supplementation.
• Gibco 2-Mercaptoethanol contains 2-mercaptoethanol at a
concentration of 55 mM in Dulbecco's phosphate buffered saline
(DPBS).

7. 1.2. JSR Autoclave
• Open the door and pour distilled water
up to the top of the cover
• Place media or laboratory equipment to
be sterilized in the autoclave.
• Close the autoclave properly and tightly.
• Turn on the power button.
• Setting the time and temperature, then
press the start button.
• After the sterilization process ends and
the temperature cools down, move the
product out of the autoclave.
Reference:
https://www.youtube.com/watch?v=prfjgtE5YVQ

8. 2.1. CELL CULTURE BASICS
Morphology of Cells in Culture
• Fibroblastic (or fibroblast-like) cells are
bipolar or multipolar, have elongated shapes,
and grow attached to a substrate.
• Epithelial-like cells are polygonal in shape
with more regular dimensions, and grow
attached to a substrate in discrete patches.
• Lymphoblast-like cells are spherical in shape
and usually grown in suspension without
attaching to a surface.

9. Flowchart of a generalized cell culture process

10. Cell Culture Equipment

11. Basic Equipment • Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)
• Incubator (humid CO2 incubator recommended)
• Water bath
• Centrifuge
• Refrigerator and freezer (–20°C)
• Cell counter
• Inverted microscope
• Liquid nitrogen (N2) freezer or cryostorage container
• Sterilizer (i.e., autoclave)
Expanded
Equipment
• Aspiration pump (peristaltic or vacuum)
• pH meter
• Confocal microscope
• Flow cytometer
Additional Supplies • Cell culture vessels (flasks, Petri dishes, roller bottles, multi-well plates)
• Pipettes and pipettors
• Syringes and needles
• Waste containers
• Media, sera, and reagents
• Cells

12. Cell Culture Hood

13. Classes of Cell Culture Hoods
Class I: protect laboratory personnel and to the environment,
but do not provide cultures protection from contamination.
Class II: designed for work involving BSL-1, 2, and 3 materials,
and provide an aseptic environment necessary for cell culture
experiments.
Class III: gas-tight, and provide the highest attainable level of
protection to personnel and the environment.

14. Air-Flow Characteristics
Cell culture hoods protect the working environment
from dust and airborn contaminants by maintaining
HEPA-filtered air over the work area.
• Horizontal flow hood: blowing parallel to the work
surface. It provides protection to the culture or to the
user.
• Vertical flow hood: blowing from the top of the
cabinet onto the work surface. It provide protection to
the user and the cell culture

15. Clean Benches
• These devices only provide product protection.
• Used for: the dust-free assembly of sterile
equipment or electronic devices
• Never used when handling cell culture
materials or drug formulations, or when
manipulating potentially infectious materials.

16. Cell Culture Hood Layout
The basic layout of a cell
culture hood for right-
handed workers.
Left-handed workers may
switch the positions of
the items laid out on the
work surface.

17. Incubator
• Purpose : to provide the appropriate environment for cell growth.
• The incubator should be: large enough, have forcedair circulation, and
have temperature control to within ±0.2°C.
• Frequent cleaning
• Types of Incubators
• Dry incubators: more economical, but require the cell cultures to be
incubated in sealed flasks to prevent evaporation.
• Humid CO2 incubators: more expensive, but allow superior control of
culture conditions. They can be used to incubate cells cultured in Petri
dishes or multi-well plates.

18. Aseptic Technique

19. Check list
• Sterile Work Area: routine
cleaning by 70% ethanol
• Good Personal Hygiene:
washing hands, wearing
protective equipments.
• Sterile Reagents and Media
• Sterile Handling

20. Youtube references
• Preventing Contamination in the Cell Culture Lab
https://www.youtube.com/watch?v=P4dTbKVDIZ4&t=2s
• Prepping and Cleaning Biosafety Cabinets
https://www.youtube.com/watch?v=HJtujdOBnMY
• Sterile Cell Culture Technique
https://www.youtube.com/watch?v=ugcGo42VNqI

21. Cell freezing Cell thawing
• High concentration, low a passage number. At
least 90% cells are viable.
• Slowly: reducing the temperature
~1°C/minute.
• The freezing medium should contain a
cryoprotective agent (DMSO or glycerol)
• Store the frozen cells below –70°C (frozen cells
begin to deteriorate above –50°C)
Notice sterile technique.
• Working quickly ensures that a high proportion
of the cells survive
• Rapidly (< 1 minute) in a 37°C water bath.
• Dilute the thawed cells slowly, using pre-warmed
growth medium.
• Plate thawed cells at high density to optimize
recovery.
• Always use proper aseptic technique. Cryovials
stored in liquid-phase present a risk of explosion
when thawed.
Principles

22. Cell freezing Cell thawing
1. Make cell stock media :
DMEM 850ul + FBS 100ul + DMSO 50ul
After mixing the above together.
2. Aspirate off the media and wash plate with 1X PBS
3. Remove PBS and add 5ml cold versine (EDTA)
4. After 2~3min, collect cells from plate, pipetting up and down.
5. Add the cell suspension to a 50ml tube containing 5ml 10% FBS
DMEM.
6. Centrifuge at 1200rpm for 5min at 4℃
7. Remove sup and resuspend cells with 5ml cell stock media. (ex)
5 vial)
8. Transfer 1ml cell suspension into each vial (cryotube)
9. Put vials in -20℃ freezer as quickly as possible
10. After -20℃, transfer vial to -80℃ deep freezer and keep there
overnight.
11. The next day, transfer vial to liquid nitrogen tank.
1. Remove vial from liquid nitrogen and
immerse immediately into 37℃ water bath until
the media is liquid (approximately 2minutes),
and constantly agitate.
2. Tighten vial cap, ethanol vial thoroughly on
outside.
3. Add 1ml of media into the vial, drop by drop.
4. Add cell suspension into 3ml of fresh media
in a 15ml tube.
5. Centrifuge at 1000rpm for 3min
6. Remove sup and add 1ml of fresh media and
suspend cell
7. Prepare the 60mm dish by adding 5ml media.
8. 37℃ incubation.
Protocols for MIN6 cell

23. 1. Remove and discard the spent cell culture media from the culture vessel.
2. Wash cells using a balanced salt solution without calcium and magnesium
(~ 2 mL /10 cm2 culture surface area). Gently add wash solution to the side of the vessel
opposite the attached cell layer to avoid disturbing the cell layer, and rock the vessel back
and forth several times.
3. Remove and discard the wash solution from the culture vessel
4. Add the pre-warmed dissociation reagent to the side of the flask; use enough reagent
to cover the cell layer (~ 0.5 mL/10 cm2). Gently rock the container to get complete
coverage of the cell layer.
5. Incubate the culture vessel at room temperature for approximately 2 minutes.
6. Observe the detachment of cell under the microscope. If cells are less than 90%
detached, increase the incubation time a few more minutes, checking for dissociation
every 30 seconds. You may also tap the vessel to expedite cell detachment.
Passaging Adherent Cells
Cell subculture

24. 7. When ≥ 90% of the cells have detached, tilt the vessel for a minimal length of time to allow the cells to
drain. Add the equivalent of 2 volumes (twice the volume used for the dissociation reagent) of pre-warmed
complete growth medium. Disperse the medium by pipetting over the cell layer surface several times.
8. Transfer the cells to a 15-mL conical tube and centrifuge then at 200 × g for 5 to 10 minutes. Note that
the centrifuge speed and time vary based on the cell type.
9. Resuspend the cell pellet in a minimal volume of pre-warmed complete growth medium and remove a
sample for counting.
10. Determine the total number of cells and percent viability. If necessary, add growth media to the cells
to achieve the desired cell concentration and recount the cells.
11. Dilute cell suspension to the seeding density recommended for the cell line, and pipet the appropriate
volume into new cell culture vessels, and return the cells to the incubator.
Cell subculture
Reference: https://www.youtube.com/watch?v=CMRKKl9XSDU
Passaging Adherent Cells

25. Cell subculture
• Subculture when 80% confluent or less. Remove and discard culture medium.
• Briefly rinse the cell layer with PBS without calcium and magnesium to remove all
traces of serum which contains trypsin inhibitor.
• Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an
inverted microscope until cell layer is dispersed (within 5 to 15 minutes).
• Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension into new culture vessels.
• Incubate cultures at 37°C
Subculture Adherent Cells

26. Counting Cells in a
Hemacytometer
1. Clean the chamber and cover slip with alcohol.
Dry and fix the coverslip in position.
2. Harvest the cells. Add 10 μL of the cells to the
hemacytometer. Do not overfill.
3. Place the chamber in the inverted microscope
under a 10X objective. Use phase contrast to
distinguish the cells.
4. Count the cells in the large, central gridded
square (1 mm2). The gridded square is circled in
the graphic below. Multiply by 104 to estimate
the number of cells per mL. Prepare duplicate
samples and average the count.

27. 2.2. Immunoassay Techniques

28. These methods are based on the protein/antigen-antibody reaction that is
shown on the left side—here is the indirect method: antigen → primary
antibody → secondary antibody conjugated with a fluorochrome or an enzyme.

29. Immunofluorescence
• A technique that uses antibodies to label protein with fluorescent
marker in order identify localization and expression level in cell (ICC)
and tissues (IHC).

30. IHC vs ICC
IHC ICC
Sample Type
- Tissue sections
- Cells are surrounded of the
corresponding tissue architecture and of
other cells present in it
- Intact cells
- All or most of the extracellular
matrix removed
Sample Source - Obtained directly from the patients
- Obtained from suspension samples
(aspirates, blood smears, swabs …) or
from cell cultures.
Sample
Processing
preservation - Frozen or paraffin-embedded - Unnecessary
fixation - Binding reagents such as formaldehyde
permeabilizati
on
- Depending on the thickness of the
sections and the method of fixation
- Must be permeabilized
( Allowing the antibodies can access
the intracellular targets)
Marking Method
- Chromogenic reagents
- Using Immunofluorescence is increasingly widespread in ICC (IF) and IHC (IHF)

31. Immunohistochemistry (IHC)
• Immunohistochemistry (IHC) uses
antibodies to detect the location of
proteins and other antigens in tissue
sections.
• The antibody-antigen interaction is
visualized using either chromogenic
detection with a colored enzyme
substrate, or fluorescent detection
with a fluorescent dye.

32. Immunohistochemistry (IHC)
Main steps

33. ELISA
Enzyme-linked immunosorbent assay
Classification
ELISA (which stands for enzyme-linked
immunosorbent assay) is a technique to
detect the presence of antigens in
biological samples. ELISA relies on
antibodies to detect a target antigen
using highly specific antibody-antigen
interactions

34. ELISA
Enzyme-linked immunosorbent assay
Comparison

35. Protein microarray
Three categories of protein microarrays.
(A) Analytical protein microarrays are mostly
represented by antibody arrays and focus on
protein detection. In this class of microarrays,
targeted proteins can be detected either by
direct labeling or using a reporter antibody in
sandwich assay format.
(B) Functional protein microarrays have broad
applications in studying protein interactions,
including protein binding and enzyme-substrate
reactions.
(C) Reverse-phase protein microarrays provide a
different array format by immobilizing many
different lysate samples on the same chip.

36. Advantages Disadvantages Best applications
ELISA
Medium specificity
High sensitivity
High quantitation
Low variability
Automation potential
Good reproducibility
False positives
High sample use
High reagent use
Time consuming
Labor intensive
Costly setup for automation
or high throughput
High throughput
screening, automation
Receptor inhibition
Relative quantitation
Ample sample availability
Protein microarray
Medium specificity
High sensitivity
Highest throughput
Low reagent use
Low variability
Low sample use
Good reproducibility
Multiplex capability
Costly setup for low
throughput, mainly costs of
scanner and high density
arrays.
False positive/negative
High throughput screening
Limited sample availability
Relative quantitation
Multiplex analysis
Proteome assessment
Screening antibody cross-
reactivity
Pathology/diagnostics
ELISA vs Protein miccroarray