Hello everyone, I'm trying to stain human blood PBMC and monocytes at day 0 and day 6 M2 macrophages (obtain after differentiation from monocytes). After FACS analysis of my cells, first I don't understand why the CD14 staining of my monocytes at day 0 is low (plot 1 + 2) , as they should be strongly positive. Moreover, why with the same voltage setting my CD14 is high (as expected, see plot 3) in M2 macrophages ? Did they increase CD14 ? But I don't understand why my CD14 is low at day 0, I don't want to increase voltage in order to have same settings between day 0 and day 6.
Thank for helping me
Dear Vincent, on first look, it does seem you are being rather conservative with the voltage for PE, the CD14+ seperation you have from PBMCs is obvious (could you share as a scatter plot too?) but you have another 3 logs to play with to make the separation better.
As for the Macrophages, do you have an unstained control for these, as these cells are notorious for very heavy autofluorescence in that channel. Samuel J Lara Reyna
Thank Charlie for your answer. Please find more images here (plot 1 = CD14 on PBMC ; plot 2 = unstained macrophages).
I'm not sure to understand, do you advice me to increase voltage only on d0 monocytes ? If I'm correct, I think I shouldn't change parameters between d0 monocytes and d6 macrophages for good comparison.
Dear Vicent!
Please You look at the article:
Article A protocol for rapid monocyte isolation and generation of si...
Fig 1. Gate strategy for flow cytometry analysis.
A, B and C = representative experiment after ficoll gradient followed by two steps of cold-aggregation and by cold-aggregation + percoll gradient for isolation of monocytes. D, E and F = representative experiment after ficoll gradient followed by Immunomagnetic beads for isolation of monocytes. G, H and I = representative experiment for monocyte differentiation to moDC. A = Dotplot of size FSC-A vs FSC-H for doublets exclusion, gated on singlets. B = Dotplot of FSC-A vs SSC-A for PBMCs, inside singlets gate. C = Dotplot of CD14 vs CD3, inside PBMC gate, where Q1 represents monocytes (CD14+) and Q3 represents T lymphocytes (CD3+). D = Dotplot of FSC-A vs FSC-H for doublets exclusion, gated on singlets. E = Dotplot of FSC-A vs SSC-A for PBMCs, inside singlets gate. F = Dotplot of FSC-A vs SSC-A inside the PBMC gate, where lymphocytes and monocytes were gated according to their size and granulosity characteristics. G = Dotplot FSC-A vs FSC-H for doublets exclusion of moDC after the differentiation protocol, gated on singlets. H = Dotplot of FSC-A vs SSC-A for moDCs, inside singlets gate. I = Dotplot of CD14 vs CD209, inside moDCs gate, where Q1 represents moDCs (CD209+) and Q3 represents non-differentiated monocytes (CD14+). All percentage and statistical analysis were performed inside the PBMC gate, thus excluding cell debris.
Thanks Alexandr, but I think my protocol for isolation is good, I just have some trouble with voltage setting or signal intensity of with my FACS... I still don't understand why.
Dear Vincent as Charlie Bridgewood mentioned monocytes and macros show high autofluorescence. It is quite hard to find the problem with little information, could you share how did you prepare your samples? This could be due to multiple reasons. Do you have your Isotype Control Antibody?
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