12 color lsrii cytometer

Single cell suspensions were prepared from lymphoid organs by mechanical disruption, and stained with a mixture of fluorochrome-conjugated anti-mouse monoclonal antibodies to the following markers: B220 (clone RA3-6B2), IgM (11/41), GL7 (GL7) (from eBioscience/Affymetrix, San Diego CA), CD19 (6D5), CD21/35 (7E9), CD23 (B3B4) (from Biolegend, San Diego, CA) and CD1d (B3B4), CD95 (JO2), CD3 (145/2C11) (from BD Biosciences, San Jose CA). Dead cell exclusion was carried out in all samples using Live/Dead fixable violet dead cell stain kit (Life Technologies/Thermo Fisher, Waltham, MA). Samples were run on either a 12-color LSRII cytometer (BD Biosciences, San Jose CA) and analyzed by FlowJo software (Tree Star Inc., Ashland, OR) or 8-colors Stratedigm S1300 and analyzed by CellCapture software (Stratedigm, San Jose, CA). Bin cells were defined as CD19⁺/B220⁺, CD23⁺CD21/35^highCD1d^high. Gates for these markers were defined for every experiment based on the marker distribution on parallel samples of spleen B cells (CD23⁺ CD21/35^low follicular B subset vs CD23^lowCD21/35^highCD1d^high marginal zone B cell subset).
In adoptive transfer experiments, B220⁺ CD23⁺CD21/35^low follicular B cells (FoB) were sorted from WT and TNFR1/2 KO mouse spleens using a Becton Dickinson FACSAria cell sorter (BD Biosciences, San Jose CA).

Bone marrow and spleen single cell suspensions were stained for dead cell exclusion using Live/Dead fixable violet dead cell staining kit (Invitrogen Life Technologies, Grand Island, NY). Surface markers were stained with a mixture of fluorochrome-conjugated antibodies, which included: CD19 (clone 6D5, BioLegend, San Diego, CA), B220 (clone RA3-6B2, eBioscience, San Diego, CA), CD138 (clone 281–2, BD bioscience, San Jose, CA), IgD (clone 11.26c.2a, BioLegend, San Diego, CA), IgM (clone II/41, eBioscience, San Diego, CA), IgG (eBioscience, San Diego, CA), MHC class II (clone 500A2, eBioscience, San Diego, CA), CD80 (clone I6-10A1, BD bioscience, San Jose, CA) CD86 (clone GL-1, BioLegend, San Diego, CA).
Fluorescence minus one (FMO) controls were included in each staining protocol and used to set specific gates (22 (link), 26 , 27 (link)). 6-peak validation beads were used for calibration during the time course analysis. Samples were run on a 12-color LSRII cytometer (BD bioscience, San Jose, CA) and analyzed by Flow Jo software (Tree Star Inc., Ashland, OR).

Single cell suspensions were prepared from lymph nodes by mechanical disruption and stained with a mixture of fluorochrome-conjugated monoclonal antibodies: CD3 (clone 17A2), CD11b (M1/70), MHC II (M5/114.15.2) and Gr1 (RB6-8C5) from Biolegend, CD19 (1D3) and CD11c (clone HL3) from BD Pharmingen and F4/80 (BM8) from eBioscience (San Diego, CA, USA) as previously described (13 (link)). All the samples were stained for dead cell exclusion using live/dead fixable violet dead cell stain kit (Invitrogen, Grand Island, NY, USA) and fixed in 1% formaldehyde before running on the instrument. Samples were run on a 12-color LSRII cytometer (BD Pharmingen, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).