Berglund:General Cell Culture Methods

PLATING/SPLITTING

1. Aspirate media
2. Wash HeLa cells with .5 volume of Petri dish plate with 1X PBS (Petri dish plate is 10mL so was with 5mL of 1X PBS)
3. Shake dish vigorously
4. Add 1mL of RT (trispin-like enzyme) to each plate and put in the 37C incubator for approximately 7 minutes.
5. Tap sides of the Petri dish to help resuspend.
6. Add 10mL of low growth medium to the dish and mix well by pipette. Transfer to a 15mL conical tube.
7. Centrifuge the conical tube for 1 minute at 1000 rpm
8. Aspirate off liquid
9. Resuspend cells in 12mLs of high growth media
10. Put 10uL in each side of the cell counter and count how many cells are in each grid. The average number of cells should be 15-20 per grid.
A perfect amount of cells is an average of 15 cells per block 9which is 1.5 *10^5 cells/mL). I have an average of 32 cells per block (double what we want) so I will plate .5mLs per well in 2mL per well of high growth medium.

11. Add 2mL of high growth medium in each well and add .5mL of cells in each well
12. In 2 Petri dishes add 8mLs of high growth media and 2mL of cells (split 1:4)
13. Put wells and plate in 37C incubator

TRANSFECTION

1. Obtain an equivalent number of 2mL eppendorf tubes to how many wells that are to be transfected
2. Label each eppendorf tube and add 500ng of each plasmid so the total per eppendorf tube is 1ug of plasmid. For example, add 25ong of pcDNA3 SERCA 21-23 and 250ng of pcDNA3 empty vector. This can be done in the lab and brought to the tissue culture room and placed in the fume hood.
3. Heat up low growth media and 1X PBS while preparing transfection
4. Set 25mLs of low growth media that was aliquoted into a 50mL conical tube and the lipotransfectamin within the fume hood.
5. Obtain two 2ml eppendorf tubes and add 250uL*number of transfections of low growth media from the conical tube to both 2mL eppendorf tubes. Example, for six transfections add 1500uL of low growth media into both 2mL eppendorf tubes.
6. Aliquot 250uL from one of the 2mL eppendorf tubes to each tube with DNA in it. Throw this tube in waste when done.
7. In the other 2mL eppendorf tube add 5*number of transfections of lipotransfectamin and invert several times to mix. For example if you are doing six reactions then add 6*5=30uL of lipotransfectamin. Then add 250uL to each eppendorf tube that contains the DNA so now each ependorf tube has ~500uL volume in it.
8. Let incubate in fume hood for 20 minutes.
9. Obtain plate and six well plate from 37C incubator and look at cells. Cells in six well plate should be between 75-90% confluent for transfection. Plate should be between 80-90% confluent to be split. The six well plate was ready to be transfected and the plate was not ready to be split.
10. Place 6 well plate, 1X PBS and bottle of low growth (that has been warmed to 37C) in the fume hood.
11. Wash the cells in the 6 well plate by aspirating off the high growth liquid, adding 2mLs of 1X PBS to each well, shake vigorously, aspirate off 1X PBS and add 2mL of low growth media to each well.
12. Put plate back in 37C until the 20min incubation period is up.
13. Put away low growth media in 50mL conical tube and lipotransfectamin in fridge. 14. After 20min incubation time is up add all 500+uls of transfection reaction into their respective well in the 6 well plate. Swirl after every time you add something to a well. Put plate in the 37C incubator for 4-6 hours

WASHING CELLS AND PUTTING IN HIGH GROWTH MEDIA

1. Place 1X PBS and high growth media in 37C incubator for 20-30 minutes.
2. After warming media above, aspirate of media from the 6 well plate.
3. Wash with 2mLs of 1X PBS, shake vigorously and aspirate off media.
4. Add 2mLs of high growth media and let incubate at 37C overnight