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Each group will need to make up 20 mL of sterile culture media. Do not open any of the sterile solutions outside of the biological safety cabinet as this will contaminate them. Also, make sure to use appropriate sterile technique when handling these solutions in the biological safety cabinet. If
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you have any questions about your sterile technique, please do not hesitate to ask your TAs or lab demonstrator. It is recommended that you watch the Lab #1 video to remind you about the principles of sterile technique before you come to your Lab #2 session. 1. A 50 mL conical tube will be located at your lab station. Label this tube with the term ‘DMEM’ and your group name. 2. Calculate the volumes of DMEM, FBS, amino acids and penicillin/streptomycin that you will need to add to you tube to make up 20 mL of culture media. Place this information in Table 1 below.  Table 1. Components of Culture Media.  Percent (vol/vol) Volume (mL) Fetal Bovine Serum 10%  Penicillin/ Streptomycin 1%  Amino Acids 2%  DMEM 87%  Total 100% 20  
3. Add each of the components listed in Table 1 to your conical tube. You must do this inside of the biological safety cabinet. Place the lid on your tube and invert it two times. Place your tube in the 37°C water bath for later.  
Measuring Cell Density and Cell Viability 1. Obtain a vial of cryopreserved C2C12 cells from your TA.  2. Immediately submerge the vial 2/3 of the way into the water bath set to 37°C. Gently swirl the vial around as the solution thaws. It should take approximately 2-4 minutes for the entire vial to thaw. Make sure that you do not let the lid of the vial come into contact with the water in the water bath as this poses a risk for potential contamination. If this does happen, wipe the vial with a kim wipe, thoroughly spray it with ethanol and allow the ethanol to evaporate. 3. Label the lid of the sterile 1.5 mL microcentrifuge tube present at your lab bench with a unique group name. Once your cryopreserved cell solution has thawed completely, go to the biological safety cabinet and transfer the contents of the vial into the microcentrifuge tube.  4. With the assistance of your TA, centrifuge the cells for 3 minutes (during this time, get your supplemented DMEM from the water bath and place it in the biological safety cabinet). 5. Discard the supernatant in the waste beaker located in the biological safety cabinet. Be careful not to disturb the cell pellet. 6. Add 1 mL of fresh culture media to the microcentrifuge tube using proper sterile technique. Gently pipette up and down to resuspend the cells. To avoid creating air bubbles, make sure that the tip of your micropipettor is constantly submerged in the media. 7. Ask your TA for a microcentrifuge tube containing 45 µL of PBS and 45 µL of 0.4% Trypan Blue. Inside of the biological safety cabinet, add 10 µL of your cell sample to this tube. By what factor did you just dilute your cells? ___________ 8. Pipette up and down to disperse your cells within the Trypan Blue solution.  Trypan Blue is a toxic and potentially carcinogenic compound that you do not want to come into contact with your skin. Make sure that you wear gloves, safety goggles and a lab coat when you handle this solution.  9. With the assistance of your TA, add 10 µL of your Trypan Blue/PBS/cell sample solution to
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a hemocytometer. Place a coverslip on the top of the hemocytometer, away from the droplet of solution. Slide the coverslip toward your solution and the solution should get sucked up by the coverslip.  10. Place the remaining 990 µL of your original cell sample into the water bath for later. 11. Place the hemocytometer on the inverted microscope. Focus on one of the secondary squares located on one of the four corners of the hemocytometer grid. You should use the 10X objective lens when counting your cells. 12. Count the number of cells that have taken up Trypan Blue into their cytoplasm and nucleus in 4 large secondary squares on the hemocytometer. ____________ cells.  Count the number of cells that have excluded Trypan Blue in the same 4 squares. ____________ cells. Try to finish counting your cells within 5 minutes of adding Trypan Blue, as extended exposure to Trypan Blue can lead to cell death. 13. Take a picture of your cells on the hemocytometer. Add a scale bar to this image and save the file in the appropriate BIOL 2P03 lab section folder on the desktop. Make a note of your file name _____________________________. You will include this image in your formal lab report on labs 2-4. 14. Calculate the percent viability of your cells using the following equation: % Cell Viability = Number of cells that excluded Trypan Blue x 100%                                               Total number of cells  15. Determine the density of cells in your diluted sample using the following equation: Cell Density (cells/mL) = # cells that excluded Trypan Blue/Volume of secondary squares. **If you counted 4 secondary squares, your volume would be 4 x 10-4 mL. __________cells/mL 16. Now determine the cell density in your original, undiluted sample by multiplying this number by your dilution factor (Which is what?). __________ cells/mL



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