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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?
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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.
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