Cell Mediums

RPMI + 1X P/S + 1X L-glu without serum (RO)  take the RPMI and dilute 1:100 with streptamycin and glutamine

Thawing Cells

Reagents: RPMI media (Roswell Park Memorial Institute) (Cellgro makes this). This is a special media for mammalian cells which have necessary vitaminnes, syrum and growth factors. 

(1) Look in log book (ex. “rack 10 box 2”) for location of cells (ex. MT-2 cells which are a cell line that express CD4+ receptor on their surface and are very susceptible to HIV infection). Mammalian cells will ideally be stored at 186 c whereas bacterial cells lines are typically stored at -70 or -80. Mammalian cells are frozen in presence of a 10% DMSO (dimethy sulfoxide). This means that mammalian cells will need to be stored in nitrogen machine. Take 2 cell tubes out. If you are not going to be working with the cells right away, put them at -80.

(2) Transfer cell tubes to ice bucket and then quickly thaw cell tubes in 37 c water bath. Do not overthaw since the presence of the DMSO can kill the cells!  You want to thaw just to the point the cells have thawed. 

(3) transfer cells using (2 ml pipette) into conical sterile tube (spray the tube and cap with 70% ETOH to help with sterility) which has 50 ml of RPMI media. 

(4) label the tube appropriately. (ex. MT-2, LN98074 F 1/19/99 T 6/16/03 JL 6/16/03)

Counting Cells

1) Look at the cells under a microscope. They will be clumped together.

2) Take a portion (~5 ml) from the flask of cells and transfer to sterile tube. Using the pipette, mix and break the cells by pipetting up and down about 10 times in the conical tube. This should be done under the hood.

3) Clean off with 70%ETOH a hemacytometer slide and cover slip with klenex. Transfer about 50 ul of the cells into a pipette from the conical tube and add 50 ul of Trypan Blue (can be purchased from Sigma) so that you end up with a 1:1 dilution.. Release the cells slowly into the groove of the hemacytometer so as to fill up the groove.

4) using a counter, count the number of live cells (they will have a shine about them) and dead cells (they will be stained by the trypan blue) in the 4×4 grid of the slide. The total volume of the grid is 0.1 ul. The total viability of cells can be determined by taking # of live cells/total # of cells * 100. For example, if you count 18 live cells out of a total of 34, this would be 18/34 * 100 = ~53 viability. In general, you want the viability up above 80%.

Question: 0.1 ul times what is equal to 1ml?

Answer: 0.1 ul x 1ml/1000ul = 0.0001 ml   So 0.0001 ml would need to be multiplied by 104 or 10, 000 to get 1 ml. This means that the total cells which we count in 0.1 ul under the microscope would have to be multiplied by 10,000 to get the total # of cells in ml of solution. So if you count 18 live cells, this means that you have 180K cells/ml. But since your are diluting in Trypan Blue at 1:1 you need to multiple this number by 2. So you would actually have 180K X 2.

Subcloning Cells

1) Cells will eventually use up their media and should be transferred to new media to maintain viability. One way to know when it is time to transfer the cells into new media is when the media becomes turbid (slight yellow color). So take a new flask and add about 35 ml of new media. To the old flash, add about 25 ml of media to provide those cells with new media. Label the flasks appropriately. Ex. MT-2, LN90741 F: 1/19/99 T 6/17/03 split 1:1 6/17/03.

Enriching Cells

(1) Count the total # of cells that you want to enrich. (ex. if you counted 24 lives cells above, you would multiply that by 10K and by 2 (for dilution factor) to get 480,000 cells/ml. Suppose your flask volume is 80ml you would multiply by 80 to get 38,400,000 cells total. Suppose you do this for a 2nd flask and get 59,200,00 cells total for 80ml in that flask. So you would add the total cells from both flasks to get 97,600,000 cells. We will want to have a final concentration of 1 * 108 cells/2ml.

97,600,000cells/(x)ml = 1 * 108 cells/2ml

x=1.952     So we are going to need to resuspend our cells in about 2 ml

(2) Prepare RPMI-5. (so if you have 20% you could add 10 ml R-20 and 40 ml R-0)

(3) Distribute cells into 50 ml tubes (yellow cap). So we could use 4 tubes with 40 ml each. 

(4) centrifuge 15 min, 800 RCF (or ~2160 RPM) (Accel=fast) (brake=slow)

(5) decant and resuspend pellet with 1 ml of solution(2) then transfer this volume to 2nd tube, and so forth until last tube where you bring the volume up to 2ml with solution(2) [cell concentration could be off so can also bring volume up to 2 ml with solution(2)]

(6) Add an equal volume of Ficol-Paque using glass pipette. Slowly release the Fico-Paque at the bottom of the tube being careful not to dispense any while moving the tube down or out. (create a balance tube having and equal volume of Ficol and RPIM-5]

(7) centrifuge again

(8) Using glass pipette suck out the fluffy layer in tube and transfer to new tube. Add  5 ml RPMI-5. 

(9) Centrifuge 10 min at 1080 RPM

(10) pour off supernatent and resuspend pellet with 10 ml of RPMI-5 

(11) resuspend pellet in 10 ml RPI-5. Check cell viability 1:5 by taking 10 mul of cells to 40 ul trypton blue.

(12) Divide cells in tube into flasks with desired volume (ex. 5 ml RPMI R-20 + 35 ml R-10. ex. 50 ml R-10)

Freezing Cells

Suppose that after counting cells (above) you count 53 live cells and 0 dead. So you would have 10,000 * 53 = 530,000 cells per 1 ml. You would actually have twice this amount or 1,060,000 cells/ ml in the flask since we diluted 1:1 with tryptan blue. 

Question: We want to take out 20 ml of our cells and resuspend those cells after centrifusion in what volume if we  want final [cell] to be 2 * 106 cells/ml? 

1,060,000cells/ml * 20 ml * = 21,200,000 cells total

21,200,000 cells/x ml = 2,000,000 cells/ 1 ml

21,200,000 / 2,000,000 = x

10.6 ml = x

(1) Obtain and label specialized cell tubes. 

(2) we want to get tubes cold to enhance freezing so place them at -20.

(3) prepare freezing media. We want a 20 ml total volume freezing media (DMSO which is dimethly sulfoxide)  . So get conical tube (yellow cap) and add 

–18 ml of FBS (since we 90% FBS (20 * .90 = 18) +

–2ml DMSA (since we want 10% DMSO)

Mix tubes by inverting. Label (ex. “FM 90% FBS + 10% DMSA JL”) and place at -20c

(4) Obtain the flask with cells that contains 100% viability and add 20 ml to a conical tube.

(5) centrifuge tube at 1100 RMS for 10 min (make sure tubes are balanced and swinging buckets in tact)

(6) pour off liquid (cells will be at bottom)

(7) Transfer freezing media (3) above to tube (6). Pipette solution up slowly with 25 ml pipette to dissolve the cells. Use 5 ml pipette to transfer 1.3 ml into 14 vials (so will be using 18.2 ml total). 

(8) Place cell tubes into freezing container which has isopropyl alcohol poured into the bottom. Place this container at -70 for 4 hours (we want to freeze cells slowly which is the exact opposite from thawing). Label the 14 tubes “B16H78 2 * 106 6/28/03 JL”.