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Autoclave PBS, cell counting, cell number calculation

fatemeh safari


Posted 4/5/2010 4:00:42 AM
Hi all,



Can anybody kindly answer my following questions:

1) How long 10xPBS should be autoclaved prior to use?



2) I've diluted 10xTrypsin with PBS to 1xTrypsin, can I store it in -20deg fridge and use it later(maybe in 2-3 days) ?



3) I did cell counting but the number of cells per squre (in hemocytometer) is too large( around 350) is that correct, or I have to dilute the cells( how)?



4) if I don't use trypan blue, how is the cell calculation?



Thanks!


samm


Posted 4/5/2010 10:47:40 AM
1) 15 min/121 dC - ensure that you do it in a pre-autoclaved bottle, and prepare it in good 18 Mho waterto minimize endotoxin contamination.

2) Yes - frozen, okay, storage at RT/refrigerator is bad since trypsin is autocatalytic.

3) Optimal range 30-300 cells in the WBS chamber (16 squares in a 4x4 grid). Dilute with PBS

4) WBC chamber count x 10e4 x dilution factor (cells/ml)
fatemeh safari


Posted 4/5/2010 10:36:07 PM
Dear Samm

Thank you.

still some questions ;)

1) for dilution, I just add PBS to my medium(which contains cells)? how can I calculate the amount should be added?

2) what is dilution factor in the calculation( is that the amount of PBS which is added?)
TheFFM


Posted 4/6/2010 12:12:26 AM
1) do a 1 in 10 dilution -



put 10 µl of cell in 90 µl PBS mix and then put 10 µl of that onto the hemocytometer chamber.  do not dilute the stock suspension of cells until you know what their density is, so that you can dilute them to what you need with media for whatever you application is after you have counted them.



if the the cells are still too dense do another 1 in 10 dilution of the original 1 in 10 dilution to get a 1 in 100 dilution and count that.



2) the dilution factor is by how many times the cells you loaded into the hemocytometer have been diluted from the original cell suspension e.g 10 or 100 fold.



so e.g



if you ended up having to dilute the cells 1 in 100 to be able to count them, the cell count will be



WBC chamber count x 10e4 x 100 cells/ml
fatemeh safari


Posted 4/6/2010 1:38:12 AM
Thanks TheFFM. now it's clear to me ;)

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