3 replies to this topic

[shaum18]

Hi all,

I'm trying to phenotype macrophages derived from PBMCs. I obtained monocytes from the PBMCs by negative selection and differentiated them into macrophages with M-CSF. I'm trying to phenotype them with CD14, CD11b and CD68. However, I'm hitting a major stumbling block when it comes to blocking getting unspecific positive staining. I use murine IgG1 as my isotype control but everytime I'm getting a positive stain for that along with my main antibodies.

I was hoping to get an idea of a good protocol which would effectively block my cells properly.

Thanks!

Shaum

P.s - My antibodies are unconjugated. its indirect staining and my secondary is alexa fluor 488

[Tabaluga]

Can you roughly tell us about your current protocol ? What are you blocking with ? Do you use BSA, serum,....
And have you tried using FcR-blocking reagents ?

[shaum18]

So, I wash them in my buffer which contains 5% fcs and 0.1% bsa and 0.01% azide and then i move to the blocking phase. I tried 10% rabbit serum, human type Ab serum with the wash solution but that didn't work. I tried miltenyi fc blocker as well. I do blocking for 5mins at room temperature. After the blocking i wash the cells again and then move to antibody incubation phases and after the last wash (after secondary antibody incubation) i fix the cells which are ready for the machine

[pesji]

Maybe you should precise a bit more, did you run a secondary antibody contrôl ? I mean cells stained with only your anti-mouse Alexa 488. The fluorescence might just come from the secondary Ab ?