Computational protocol: Antigen presenting capacity of murine splenic myeloid cells

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Protocol publication

[…] Methods used for antibody staining and flow cytometry for analysis of cell surface marker expression have been described previously [, , ]. Prior to antibody staining, non-specific antibody binding to cells was inhibited by absorption of anti-CD16/32 (FcBlock: Biolegend: San Diego, CA, USA) used at 5 μg/106 cells in 1 mL of FACS buffer. Fluorochrome- or biotin-conjugated antibodies specific for CD11c (N418), CD11b (M1/70), CD8 (53–6.7), CD19 (1D3), CD43 (IBII), F4/80 (CI:A3-1), Ter119 (Ter119), Thy1.2 (30-H12), Siglec-F (E50-2440), Ly6C (HK1.4), Ly6G (1A8) and I-A/I-E (M5/114.15.2) were purchased from Biolegend. Antibodies specific for CD68 (FA-11) and SIGN-R1 (ER-TR9) were purchased from AbD Serotec. Lastly, antibody specific for MOMA-1 (MOMA-1) was purchased from AbCam. Propidium iodide (PI) staining prior to flow cytometry was used to distinguish live and dead cells. Flow cytometry was performed on a BD LSRII flow cytometer (Becton Dickinson: Franklin Lakes, NJ, USA). Data were collected in terms of forward scatter, side scatter and multiple fluorescence channels. BD FACSDiva Software (Becton Dickinson) was used to acquire data and analysis post-acquisition employed FlowJo software (Tree Star: Ashland, OR, USA).For sorting, cells were stained with fluorochrome-labelled antibodies and subsets identified as described in Hey et al. [] and summarised in Table . All incubation and washing steps were performed in sodium azide-free FACS buffer. Sorted populations were collected in complete medium for use in functional assays. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus