Spleen, PPs and mLN cell suspensions were generated by mashing the organs through 70-μm cell strainers. For DC isolation, PPs and mLN were digested with 1.6mg type II collagenase (Worthington Biochemical) and DNase I for 10min at 37°C. Digested PPs were mashed into single cell suspension through a 70μm cell strainer in PBS buffers containing 2% FCS and 2mM EDTA. Cells were stained with Abs to CD4 (GK1.5), B220 (RA3-6B2), CD19 (1D3),IgD (11-26c.2a), CD95 (Jo2), GL7, CD38 (90), CCR6 (140706), CD8 (53), MHCII (AF6-120.1), IgA (1040-09), IgG1 (RMA1-1), CD11c (N418), CD11b (M1/70), CD45.1 (A20), CD45.1 (104) (from Biolegend, BD Biosciences, rnBiotech or eBioscience). Biotin conjugates were detected with streptavidin Qdot605 (Invitrogen). To detect intracellular IgA, cells were stained with fixable viability dye (eFluor780; eBioscience) to exclude dead cells then stained for surface antigens, treated with BD Cytofix Buffer and Perm/Wash reagent (BD Biosciences), and stained with anti-IgA antibody.
Cytofix buffer and perm wash reagent
Manufactured by BD
BD Cytofix Buffer and Perm/Wash reagent are laboratory products used for sample preparation and processing. The Cytofix Buffer is used for fixation, while the Perm/Wash reagent is used for permeabilization and washing of cells during flow cytometry or other immunological analyses.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using cytofix buffer and perm wash reagent
1
Isolation and Characterization of Dendritic Cells from Murine Tissues
2
Immune Response to Microbial Challenges
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Mice were challenged with 2 × 107 CFU of heat inactivated C. albicans, attenuated Yersinia pestis, or 150 μg S. aureus bioparticle (Invitrogen, Cat S2859) through the footpad. Control mice were treated with 25 μl saline. Draining popliteal LNs were dissected and analyzed 3 hr post challenge. For IL17 staining, popliteal LN cells were incubated in Golgi plug for 2 hr at 37°C, stained for surface antigens, treated with BD Cytofix Buffer and Perm/Wash reagent (BD Biosciences), and stained with anti-IL-17A.
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