Journal: STAR Protocols
Article Title: Protocol for isolating cervico-vaginal fluid cells from Macaca mulatta to study immunological and functional changes during pregnancy
doi: 10.1016/j.xpro.2025.103927
Figure Lengend Snippet: Gating strategy for flow cytometry phenotype analysis of CVL cells (A) Filter the CVL cell solution in a FACS 5 ml polystyrene round-bottom tube with cell-strainer cap. (B) After centrifugation, the cells pellet is visible. (C) After incubating the cells with the Ab mix, add 500 μl/tube of cold FACS washing buffer. (D) Representative density plots of Rhesus CVL samples. Forward scatter area (FSC-A) and forward scatter height (FSC-H) gating on cells to exclude doublets. Dead cells were excluded by Live/Dead Fixable Aqua Dead Cell Stain kit. FSC and side (SSC) scatter gating on cells to exclude cellular debris. CD45 − cells gate representing non-leukocyte cells and CD45 + cells positive gating representative leukocyte cells. Inside the CD45 + cells, the leukocyte subpopulations were gated as monocytes/macrophages (CD3 − HLA-DR + CD19 − CD20 − CD14 + ); activated B cells (CD3 − HLA-DR + CD14 − CD19 + CD20 + ); neutrophils (CD3 − CD14 low HLA-DR − CD88 + CD56 − ); NK cells (CD3 − CD14 − HLA-DR − CD56 + ); resting B cells (CD3 − HLA-DR − CD14 − CD56 − CD19 + CD20 + ); T cells (CD14 − CD56 − CD3 + ); and NKT cells (CD14 − CD3 + CD56 + ); myeloid dendritic cells (mDCs, CD3 − CD14 − HLA-DR − CD56 − CD19 − CD20 − CD123 − ); plasmacytoid dendritic cells (pDCs, CD3 − CD14 − HLA-DR − CD56 − CD19 − CD20 − CD123 + ). A similar gating strategy can be used to characterize human cells, with the only difference that CD66b should be used instead of CD88 for neutrophils. (E) Example of data analysis: frequency of neutrophils in CVL at different time points during gestation (before E. coli injection = gestational age 140 days; delivery = gestational age gestational age 146 days).
Article Snippet: Anti-human CD88 (clone MCA2059A647T) – dilution 1:10 , Bio-Rad , Cat# MCA2059A647T, RRID: AB_1102420.
Techniques: Flow Cytometry, Centrifugation, Staining, Injection
Miltenyi Biotec is a verified supplier 
Miltenyi Biotec manufactures this product 
