I have always labeled cells with CFSE after detaching them from the culture plate. However I need to label plated macrophages this time. What I usually do is use the required concentration of CFSE on my cell pellet and incubate at 37 for 15 minutes, followed by a room temperature 10 minute incubation in dark. I then inactivate the dye by adding medium containing 10% FBS and give it 3 washes with centrifugation at 295g. I was planning to do the same with the attached macrophages I have, but instead of the centrifugation, I was planning to wash them well using a pipette. Later I could scrape them off and read them on the flow. Any thoughts? Thank you.
You plan sounds good. the only thing I need mention is that you should use plenty of dye solution to cover all your cells. You might lose some cells if they don't attach very well.
Your protocol should work fine on the plate. Just make sure that you remove all potential sources of amine groups before you start labeling. So, probably 3X PBS washes before CFSE addition (especially if FBS was present); label; then 3X PBS washes to remove excess of the dye. I never did quenching with FBS, but it would not hurt to do it either.
Please don't scrape the cells off if you are using them in down stream flow, you will kill a large population and likely damage most of the cells that are left, I'd recommend using a gentle disassociation reagent such as accuatse or another stem cell dissociation reagent. I've found scraping cells just leads to dirty flow downstream.
Hi Samantha,
Thank you for your input. I can try using TrypLE for cell dissociation. I have used it in the past and it seems to work fine.
Manasi
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Immunology Techniques