2 replies to this topic

[leelee]

Hi all,

Sorry if this has been covered in previous posts- I can't seem to find the right search terms to locate them.

Anyway-
I'm staining my infected fibroblast cell line with an antibody against a viral protein. As this is obviously intracellular, I'm permeabilising my cells with 0.05% Tween 20 (in PBS with 2% NCS).

I find that after resuspending the cells in the perm buffer, they become very sticky and tend to clump together, making them difficult to resuspend as a single cell suspension.

Does anyone know if there is anything I can do to minimise or eliminate this problem- or is it just what happens once cells have been permeabilised?

Thanks!

Edited by leelee, 19 June 2011 - 07:32 PM.

[leelee]

Oh, and my supervisor has suggested to pass the cells through some gauze before running through the flow- but as I have a large number of samples, I'd rather avoid this if possible as it is very time consuming.

[emilytxz]

I have not stained intracellularly for fibroblasts, but usually for intracellular staining (of leukocytes) we permeabilise using cytoperm/cytofix (which you can buy from BD Biosciences) and then stain in your normal staining buffer supplemented with 0.1% saponin and 0.1% formaldehyde.