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[cellwork]

Hello,

 

I would to ask if anyone knows whether the volume of buffer I resuspend a pellet in affects cell yield.

 

Example:

 

Scenario 1 : I resuspend my cell pellet in 50uL PBS (let's say there is some antibodies I am trying to incubate the cells with). Then I wash with 100uL PBS and centrifuge at a final volume of 150uL.

 

Scenario 2: I resuspend my cell pellet in 100uL PBS (incubate with antibodies) . Then I wash with 200ul PBS and centrifuge at a final volume of 300uL.

 

Scenario 3: I resuspend my cell pellet in 100uL PBS (incubate with antibodies) . Then I wash with 300ul PBS and centrifuge at a final volume of 400uL.

 

Scenario 4: I resuspend my cell pellet in 200ul PBS (incubate with antibodies). Then I wash with 300ul PBS and centrifuge at a final volume of 500uL.

 

 

 

In which scenario will I have greater cell yield and better pellet quality after centrifugation step is complete? I would like to lose as few cells as possible. I would like to know how people determine the buffer volume they resuspend their pellet in and the volume of buffer used for washes. I have noticed in multiple papers they go for higher volumes. Does higher volumes mean better cell yield ? I need to retain as many cells in the pellet as possible and lose few as possible during the wash and centrifugation. I think the volume you resuspend the pellet in and the volume in the centrifuge matters and I would ask your opinions on the best scenario to keep as many cells as possible.

 

 

Thank you.

[bob1]

The volume doesn't matter in general, other than that you need to have a bigger volume than your cells take up. The return from centrifugation should be the same no matter how big the volume is, so long as you use sufficient time and RCF to pellet the cells. You will lose some stuck to the walls of the tube, but those will be residue from the pellet, not a function of the wash volume. You do need enough wash volume to be able to remove it efficiently.

 

The re-suspension volume is about washing the cells, so the bigger the volume you use there, the more dilute your reagent (antibodies in your case) will be and the more efficient the wash will be at diluting the reagent to a minimal concentration, meaning you can use fewer wash steps to "completely" remove the reagent.