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.
What I could get from your question is;
1• volume of buffer to stain cells with antibodies?
2• volume of buffer while washing?
To answer your question 1; usually antibody staining is done in 1*10^5 cells/100uL of cell suspension. It's standard. If you want to save antibody you may optimize appropriate volume.
Answer to question 2;
U need to wash your cells twice with buffer at least 10 times higher than that of the volume of cell suspension (1000uL) to completely remove the unbound antibodies. This is critical.
There is always loss of cells in each centrifuge, but washing in 20 or 2 mL will not affect the loss of cells. It is the centrifuge with cause it, weather in 200uL or 2mL.
I hope you got the answer.
Washing volume is much necessary for washing off the unwanted from sample.
And I found no much (remarkable) difference in washing with 300 uL to 2 mL. Increasing g force will help you reduce cells but I am not sure reducing wash volume will affect much.
Adding FBS/BSA in PBS/wash buffer (aka staining buffer) if supposed to be good as it increasing the viscosity of buffer leading to reducing mechanical forcing during centrifugation. Cells does not get stressed in staining buffer.
(by general rule you need to bare in mind in the volume you suspend. It is true the larger the better, but it is not always the case. It is determined empirically by the washing steps. It is also important what happens to the pellet. As you "purify" the washing steps gain importance and is why the pellet is more precise. Make sure you don't rip the pellet off the bottom. Otherwise you cannot make use of the washing steps. Take a look at the protocol,but it may be roughly the same as a standard.)
It is true the more pellet the better. The more cells the better and make sure the washing buffer reaches for all the eppendorffs you are using.
It would be very difficult for me to decide, but it is very difficult to compare as there are antibodies. (they had to be different)
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