I'm having issues with the stability of my CD45 stain conjugated with APC. I'm staining PBMCs for Circulating tumour cells. If I do not do the intraceulluar (CK) staining and simply CD45 stain and fix the cells the staining is stable for a few days. If I do my entire protocol which includes a CD45 stain (1 hour 4 degrees) , fixing (2% PFA for 20 mins), perm (0.1% triton for 15 minutes), intracelllular stain (1 hour, room temperature) the Cd45 stain is not visible when imaging. Any thoughts on what could be causing the quenching? We use AutoMacs running buffer for washing in between steps.
Dear Melanie,
The truth is after fixation/permeabilization the cells look different in Flow Cytometry, also due to increased autofluorescence. but that doesn't really answer to your issue.
I used BD Cytofix/Cytoperm buffers for my assays due to optimized solutions they offer. I was fixating for 20min at 4oC, then washing with the saponin solution for completely removing the PFA and staining in the same saponin solution for 30min at 4oC in the dark and wash with FACS buffer. Their fixating solution contains also saponin.
I believe permeabilizing the cells with Triton-X for too long in RT may affect the intracellular staining, as you might harshly disrupt the cell membrane and release the desired proteins or even completely lyse the cells. Prolonged times may be used for intranuclear staining though.
MACS running buffer contains EDTA, which I don't think will affect the Ab binding to the protein.
Finally, if you activate the cells with PMA/Ionomycin prior the intracellular staining, I have observed some extracellular markers to be downregulated (CD3 (mouse), TCRab (mouse), CD4 (pronounced in human, not in mouse), but not CD45, at least in mouse, but I don't know about human T cells in that case).
In conclusion, I would try different staining methods and/or fixating. Try to incubate at 4oC. Some antibodies do not react perfectly after fixation and permeabilization, for some cases fixation and permeabilization with cold pure methanol work even better, but unfortunatelly I don't have such an experience on that.
I hope you'll get "fixed" that soon!
Best,
Filip
Filippos Theofilos Charitidis Thanks! It seems to be the perm step, Saponin (30 mins, 4 degrees) seems to allow for more stable staining. Thanks again for the insight!
- Badges
- Science method