Hi,
I need assistance in planning an efficient panel for staining PBMCs in a clinical trial.
My plan is to evaluate their activation profile by using FACS analysis after ex vivo stimulation.
I've been offered to use Miltenyi's Human Blood Dendritic Cell Enumeration Kit, but I'm only interested in myeloid-derived dendritic cells and a small fraction of monocytes.
I'm still trying to figure out which fluorochromes will fit best without any spillover and loss of resolution. So this is what I've come up to-
So this is what I've come up to-
CD1c APC
CD14 FITC
CD141 PE
CD40 Pacific Blue
B7-H4 PE/Cy7
CD19 PerCP or Alexa Fluor 647
I will be using FacsCalibur (3 lasers 407nm,488nm,637nm) and the FACS Canto 2.
Any kind of help would be greatly appreciated,
Thanks!
What exactly to you mean with "myeloid dendritic cells"? Every dendritic cell is part of the myeloid lineage, and if you would like to look for DCs you definitely need at minimum CD11c and HLA-DR as markers in your panel. CD1c as well as CD141 will be marking cDC2 and cDC1, respectively, but they wont help if you dont have CD11c/HLA-DR as well.
If you are going to use a FACSCanto 2 with the standard 3-laser setup (to best of my knowledge there exists no FACSCalibur with three lasers and 8 detectors), then I would recommend you the following:
CD11c-APC
HLA-DR-APCCy7
CD14-PerCPCy5.5
CD40-BV421 (brighter than PacBlue)
CD1c-PE
CD141-FITC
Live/Dead-Aqua
B7-H4 PECy7
You might want to consider using PerCPCy5.5 as a dump channel and throw in together with CD14, CD3, CD19 and CD56.
Good luck with your stainings!
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