Single cell suspensions were generated from murine tissues of C57/BL6 and BALB/c mice. Afterwards, cells were treated with FcR-blocking reagent (Miltenyi Biotec) for 10 min at 4°C after washing with flow cytometry buffer. To exclude dead cells, samples were washed twice with PBS and incubated with BD Horizon™ Fixable Viability Stain 510 viability dye (for 10 min at RT in the dark). Subsequently surface staining to discriminate immune cell populations with PerCP anti-CD45 (30-F11, BD Biosciences), APC-Cy 7 anti-TCRβ (H57-597, BioLegend), Pe-Cy7 anti-CD11b (M1/70, BD Biosciences), and anti-CD19 (6D5, BioLegend), was performed. The data was acquired using a BD FACS LSR II instrument (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc, version 9.9.6 or 10.4.1).
Pe cy7 anti cd11b m1 70
Manufactured by BD
The PE-Cy7 anti-CD11b (M1/70) is a fluorochrome-conjugated antibody that binds to the CD11b antigen. CD11b is a cell surface marker expressed on myeloid cells, including monocytes, macrophages, and granulocytes. This antibody can be used in flow cytometry applications to identify and analyze these cell populations.
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2 protocols using pe cy7 anti cd11b m1 70
1
Murine Immune Cell Profiling
2
Fluorescent labeling of BMDCs
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BMDCs were labeled using the following commercially-available reagents: PE anti-CD11c (N418; Biolegend, San Diego, CA), PE-Cy7 anti-CD11b (M1/70; BD Bioscience, San Jose, CA), Pacific Blue anti-MHC II (M5/114.15.2; Biolegend), biotin anti-CD86 (GL1; BD pharmingen, San Diego, CA). Streptavidin-Alexa Fluor 700 (Invivogen) was used as a second step reagent. Data were collected with FACS Diva-driven LSR Fortessa (Becton Dickinson) and analyzed with the FlowJo software (Tree star, Inc., San Carlos, CA). After positive selection of the whole CD11c + CD11b + BMDC population, the frequency of CD86hi MHCIIhi BMDCs was used as a surrogate for activation (Fig. 1A). Fluorescence minus one (FMO) controls were performed at every experiment. When multiple experiments were pooled, data were normalized to an internal control (100 ng/ml endotoxins, Figs. 1,4,5). Time-course experiments (Fig. 2) show raw frequencies of CD86 hi MHC II hi BMDCs out of the total viable BMDC pool.
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