I would like to check proliferation of human CD8 cells (isolated from blood ) after few days of culture. I have a panel of antibodies and I want to add KI67 to analyse proliferation. Does anyone has a good experience with a particular fluorophore? Should I choose a bright fluo or on the contrary a dimmer color?
Many thanks for your help.
Laure
Hi Laure:
The multicolour Ab panel for FACS is a bit tricky, need some expert to help you. Generally, (1) you need a good FACS machine equipped with at least 3 lasers for your 7 colour FACS, such as Fortessa ; (2) seek help some FACS expert to design your Ab panel; (3) include biological controls, techique controls, BDcomp beads, FcR blockage into your work;
Talk to your FACS manager may be helpful.
Ki67 express in G1, S, G2 and M phases cells, not in G0, they expression amount in CD8 cells depending on the sample source, culture conditions and Abs potency.
Good luck
Shiqiu
Well I use Alexa-Fluor for both ICC and FACS. The two reasons one should use a fluorophore with a high quantum yield that I can think of ATM are (a) high difference from autofluorescence and (b) better differentiation between expression levels in cell populations (c) detection if low expression levels of Ki67. It will make a world of a difference if the difference between control and treatment is marginal.
Although I am not aware of the argument of using a dimmer fluorophore yet.
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