Index
MTT Viability Test
Objectives
·
Develop linear relationship between absorbance and cell
concentration.
·
Familiarity with theory and use of spectrophotometer.
Materials
TC-treated 24-well plates
Fibroblasts cells (HDF)
DMEM with 10% serum and 1% antibiotics
Trypsin
MTT Dye Kit: Dye solution, Solubilization/Stop solution
Foil
Micropipette
PBS
Cuvettes for spectrophotometer
Coulter Counter vials and Isoton
Materials as required by cell passaging procedure.
Safety
1. The MTT Dye Solution is hazardous. Avoid contact with skin and eyes.
2. The MTT Solubilization/Stop Solution is an organic solvent. It must be handled in the organic fume hood.
3. Wear disposable latex gloves at all times
4. Wear safety glasses at all times.
Pre-Lab
1. Design configuration of 24-well plates for assay. Test conditions include:
A. Cells Treated with MTT Dye.
· Six cell concentrations (samples) to be tested.
· Stock cell concentration is 50,000 cells/mL.
· Cells to be diluted 1:1.5, 1:2, 1:3, 1:6, and 1:12.
|
Sample |
Dilution |
Cell Concen. |
|
1 |
stock |
50,000 cells/mL |
|
2 |
1:1.5 |
33,500 cells/mL |
|
3 |
1:2 |
25,000 cells/mL |
|
4 |
1:3 |
16,700 cells/mL |
|
5 |
1:6 |
8,330 cells/mL |
|
6 |
1:12 |
4,170 cells/mL |
· Dilution stated as 1:2 is a 50% dilution (i.e. 1 unit in 2 units total).
B. Cells Counted on Coulter Counter.
· Six cell concentrations (samples) tested.
· Stock cell concentration is 50,000 cells/mL.
· Cells to be diluted 1:1.5, 1:2, 1:3, 1:6, 1:12. (See chart above)
C. Control wells that include no cells.
2. Calculate the volume of cells and volume of media required to achieve the above dilutions. The desired volume in each TC well is 0.5 mL.
3. Lab partners should set up plates together. Put test conditions for both partners for “Cells Treated with MTT Dye” on one 24-well plate. Put test conditions for both partners for “Cells Counted on Coulter Counter” on a second 24-well plate. Appropriate control wells should be set up on each plate.
Seed cells (Day 1)
1. Check for HDF cells remaining from the “Fn Attachment Assay” or from the “Live/Dead Fluorescence Assay.”
2. Confirm that HDF cells are in DMEM with 10% serum and 1% antibiotic.
3. Store cells on ice until you are ready to use them.
NOTE: Each partner should do steps 4. - 10. independently.
4. Prepare three samples, each with 500 mL of cells and 9.5 mL Isoton, to determine the cell concentration. First, aliquot Isoton into Coulter Counter vials. Place vials in laminar hood. Add cells to vials.
5. Determine the cell concentration using the Coulter Counter. Remember to correctly account for dilutions and Coulter Counter sampling volume.
6. Dilute part of your cell suspension in DMEM with 10% serum and 1% antibiotic to a concentration of 50,000 cells/mL. (If your cell concentration is already below this, do not dilute further.) This sample will be the stock concentration. Prepare a sufficient, but not excessive, volume of cells at 50,000 cells/mL (e.g., 6 mL should be enough).
7. When seeding, mix the cells gently and often (every 6 aliquots or every 15 sec) to prevent settling of the cells.
8. Seed 0.5 mL of this cell suspension into each stock test well on each of the TC-treated plates (one plate for “Cells Counted on Coulter Counter”; one plate for “Cells Treated with MTT Dye”).
9. For the wells requiring a dilution, add media and cells, as needed, to achieve the proper proportions. The total volume in each well should be 0.5 mL. (Remember: one plate for “Cells Counted on Coulter Counter”; one plate for “Cells Treated with MTT Dye”.) For example, for the 1:2 condition, add 0.25 mL cells and 0.25 mL DMEM.
10. Place cells in incubator for two nights. Cells will firmly attach to the surface during this time.
Conduct MTT assay (Day 3)
NOTE: One partner can do steps 1. - 4. for both partners.
1. Remove plate designated for MTT Dye from incubator. Look at the cells, estimate the confluency in each well, and record your observations.
2. Add 75 mL of the Dye Solution to each well designated for the MTT Dye test and the appropriate controls. Caution: Dye Solution is an irritant; avoid contact with skin and eyes!
3. Carefully wrap plate with foil. The foil should be loose enough to permit air circulation but cover the whole plate to minimize exposure to light. NOTE: The dye is light sensitive. Failure to keep the cells covered will result in errant data.
4. Place plate in incubator for 2 hrs.
NOTE: For steps 5. - 18., each partner should do her/his own work.
5. Prepare CC vials each with 9 mL of Isoton. Prepare one vial for each well containing cells (and controls) to be counted using the Coulter Counter.
6. Remove plate designated for Coulter Counter Cell Count from incubator. Look at the cells, estimate the confluency in each well, and record your observations.
7. Using a sterile glass Pasteur pipet, aspirate the liquid media covering the cells and control wells for the Cell Count test.
8. Add 1 mL PBS to each well. Swish around.
9. Aspirate liquid from each well.
10. Add 250 mL of trypsin to each well.
11. Place plate in incubator for 5 min.
12. Remove plate from incubator. Tap side of wells several times. Be careful not to spill the wells. Check cells under microscope to confirm that cells are detached from the surface.
13. Add 750 mL complete media to each well.
14. Do the following for each well, one at a time:
a. With micropipette set at 900 mL, suck the liquid up and down three times.
b. Aliquot 900 mL of cell mixture to a Coulter Counter vial with Isoton.
c. Add 900 ml Isoton/cell mixture back into the well.
d. Repeat steps a) and b).
e. Repeat steps c), a), and b).
f. Remove remainder of liquid in well to Coulter Counter vial.
15. Repeat for each well of the Cell Number Coulter Count test and Control.
16. Using a microscope, confirm that virtually no cells remain in the wells.
17. Count cells using Coulter Counter. Since cell number will be low, it is recommended that you run each sample in triplicate.
18. Dispose of biohazardous waste appropriately.
NOTE: One partner can do steps 19. - 20. for both partners.
19. Following the 2 hr incubation of cells with MTT dye, add 500 mL of the Solubilization/Stop solution to each well. Caution: The Solubilization/Stop solution contains an organic solvent. Therefore, this operation must be conducted in the chemical fume hoods (i.e. NOT the tissue culture hoods).
20. Recover the plate with foil. Leave plate in the organic fume hood for 45 min.
NOTE: For steps 21. - 23., each partner should do her/his own work.
21. Following the 45 min incubation of cells with the Solubilization/Stop solution, carefully mix the liquid in each well and transfer to a plastic spectrophotometric cuvette. Avoid bubble formation, as bubbles may interfere with the accurate reading of the absorbance values on the spectrophotometer.
22. Record the absorbance at 570 nm on the spectrophotometer. See Spectrophotometer Instructions (p. 39).
23. Dispose of all liquid that has been in contact with Solubilization/Stop solution in labeled Organic Waste Jar in hood. Liquid needs to be recovered from spectrophotometer cuvettes and 24-well plates. Dispose of biohazardous waste appropriately.
Calculations/Questions
1. (15 pts) Plot your data in a meaningful way.
2. (15 pts) Develop a mathematical relationship between absorbance and concentration of cells. Is your relationship linear? How “good” is your fit? Characterize your fit using an R2 value.
3. (10 pts) Why is running a control important?
4. (10 pts) What are some advantages of using the MTT assay (or any metabolic assay) over direct cell number (e.g. Coulter Counter)?
5. (10 pts) Propose a method/experiment to develop a relationship between absorbance and concentration of VIABLE cells.
Data and observations should be recorded in your laboratory notebook. The point values for the Calculations/Questions are listed above.