Hi. everyone!
Does anyone here familiar with indirect staining of intracellular proteins. Trying to stain106 cells, however, I only got ~500 cells. 90% cells loss :(
Do any tricks to avoid the cell loss?
(Increase to 3x106 cells, higher centrifuge force, etc)
Procedure:(all steps are proceeded in 1.75mL eppendorf)
106cells→fix→wash(~104cells)→perm→1'ab→washx2→2'ab→washx2→flow cytometry analysis (
Hello Jyun-Hong Lyu,
I had the same problem while I was trying intracelular staining. Here are some tips I found useful:
1)Try to increase centrifuge speed 1.5-2 fold more than regular centrifuge speed you use after the fixation step. ex: I am working with stem cells, regular centrifuge is 900 rpm. I increase this to 1200 rpm after fixation since this is the most critical step you lose cells.
2)Try to avoid most of the washes. It is increasing the cell loss.
3) Use the same tip for same cell line until you finish the process. Take an aliquot of the liquid into a new eppie and use same tip.
I hope it would be helpful to you.
Hi Jyun-Hong Lyu,
You lost most of the cells after permeabilization. I think you should try other permeabilizer like saponin. TritonX-100 is very strong/harsh (compared to tween20 and saponin). Cell loss might not be due to other steps like washing.
Try permeabilize cells with 0.1% (w/v) saponin (Sigma Aldrich-S7900) for 15 mins at room temperature (in the dark if surface staining has been done). Saponin in solution is only stable for a maximum of 3 days when stored at 4'c. So, I usually prepare on the experiment day. The antibody must be diluted in saponin-PBS because the membrane permeabilization is reversible.
I fix cells with 4% (w/v) paraformaldehyde-PBS for 20 mins at room temperature (in the dark if surface staining has been done).
1) If you don't have protein in the solution when you centrifuge the cells, they will tend to stick to the tube walls. Add 0.5% BSA to your first wash after fixing and to the later PBST washes.
2) After fixing, you can use higher centrifugation speed as Aysegul mentioned. Maybe 2000xg.
3) For each centrifugation step, spin the tubes for half the time with the hinge facing the center of the rotor. Then turn the tubes 180 degrees so that the hinge faces the outer rim of the rotor and spin for the remaining time. This helps the cells to form a nice pellet instead of sticking/smearing down the tube wall.
Another way to avoid cell loss during washing steps is to replace the pipette aspiration step with a split centrifugal force device like the one described here
http://www.siliconbiosystems.com/vrnxt
This allows you to remove 99% of supernatant from a pellet, or even from just one single cell, without losing any cell. in just 10 seconds.
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