Hello, I am using thp-1 cells on a cyclic olefin support for confocal microscopy. After. 2% formaldehyde staining , three washings,1% bsa and I stain them with Cell membrane staining CellMask Deep Red 1:1500 in D-PBS for 10 minutes followed by 3 washings.
However I see a lot of aspecificity of this staing, like circles of red with no nuclei, or random smears and the borders of cells are not defined.
How can I improve this? Do you have good alternatives for staining?
Thanks
Fabiola
Hi Fabiola Vacca
I guess you are obtaining well spread & adherent THP-1 cells using 50 nM PMA treatment overnight. Once they are attached you can wash them once to remove the dead/floating cells. Then you can follow the fixation protocol using 4% PFA for 20-30 mins. Post-fixation washing is equally important. Sometimes single efficient washing is better than 3 washes. The red dots you are getting might be residual dye. Also, if you feel that the cellular uptake is not good enough the incubation time with CellMask can be increased to 15-20 mins. Hope this improves the staining.
- Badges
- Science topic
- Similar topics
- Engineering
- Chemical Engineering
- Membranes