I isolate monocytes and macrophages from tissues. I need to stain surface markers, viability dye and phagocytosis at the same tube. What kinds of phagocytosis kits and viability dyes can I choose when doing flow cytometry?
In the past a researcher in our lab did a phagocytosis test using fluorescent beads. Another possibility is using E.coli expressing a fluorescent marker.
If you want to quantify phagocytosis of any kind of bacteria you can stain it by FITC. or just stain a latex beads by FITC or other fluorochrome, the inconvenient is that your dye loose signal fastly . you can choose a fluorochrome that is flurescent only in acidic pH (lysozome). And finally you can labell your surface markers with any other dyes. to contrôle viability you may need another dye.
Most protocols mention to put the phagocytes in the culture plate/well and then add fluorescent beads. I find that macrophages are hard to harvest when putting them in the culture plate/well. I have to use cell scrapers or trypsin to harvest them. I am afraid I will lose some antigens expressed on macrophages. Could I put the macrophages and fluorescent beads in the water bath and then do further surface staining? Thanks.
Dear Ya-Ping You are using a too strong way to detach cells. Use PBS-2 mMEDTA 0,5 % gelatin cold to detach the cells, then wash them with the same buffer room temperature. For the phagocytosis assay simply use the standard protocols for flow cytometry which is use FITC grown cultures of E coli and incubate a relation of 1000 to 10000 bactera per macrophage in a water bath at 37 degrees for 30 min in which the cells are resuspended in PBS 0.1 mMEDTA 0.5 % gelatin. The extracellular bound bacteria can be subtracted in the assay using Trypan blue. Good luck
We use a modified method that has been developed years ago by Valet et al that uses Dihydrorhodamine 123, which is quite successful. Look for the article in Methods in Molecular Biology Cytometry issues in 1990's.
CD68 as surface marker, Annexin-V for apoptosis/viability, opsonised Latex-dye for phagocytosis. Each conjugated with different dye. Do not forget the washing after the stain.
Dear Shamila D. Alipoor,
Thanks for your recommended manuscript. It helps me a lot.