Cd21 fitc

Subpopulations of B cells (CD3-CD19+) were analyzed by flow cytometry after the staining of surface markers CD10, CD27, CD20, and CD21 [12 (link)]. These cells include immature or transitional cells (CD10+ CD27-), naïve B cells (CD10-CD27-CD21high), tissue-like memory cells (CD10-CD27-CD21low), resting memory cells (CD10-CD27+CD21high), activated memory cells (CD10-CD27+CD21low), and plasmablasts (CD27++CD20-CD21low). Antibodies CD3-PE, CD10-BV421, CD19-BV711, CD20-AlexaFluor700, CD21-FITC, and CD27-PercP-Cy5.5 were purchased from BD Biosciences (San Jose, CA, USA). Data acquisition was performed in BD LSRFortessa X-20 flow cytometer with FACS Diva software (BD Biosciences, San Jose, CA, USA). FlowJo software (Tree Star Inc., Ashland, OR, USA) was used for data analysis.

B-cell subpopulations (CD3⁻CD19⁺) were differentiated via flow cytometry after staining the surface markers of CD10, CD27, CD20, and CD21 as follows: immature or transitional cells (CD10⁺CD27⁻); naïve B cells (CD10⁻CD27⁻CD21^high); tissue-like memory cells (CD10⁻CD27⁻CD21^low); resting memory cells (CD10⁻CD27⁺CD21^high); activated memory cells (CD10⁻CD27⁺CD21^low); and plasmablasts (CD27⁺⁺CD20⁻CD21^low). Antibodies CD3-PE, CD10-BV421, CD19-BV711, CD20-AlexaFluor700, CD21-FITC, and CD27-PercPCy5.5 were purchased from BD Biosciences (San Jose, CA, USA). Data acquisition was performed in a BD LSRFortessa X-20 flow cytometer with FACS Diva software v7.0 (BD Biosciences, San Jose, CA, USA). FlowJo software v10 (Tree Star Inc., Ashland, OR, USA) was used for data analysis.

The antibodies used were CD19-PECy5 (HIB19, Biolegend), CD56-APC-Cy7 (clone HCD56, Biolegend), CD21-FITC (Clone B-ly4, BD Pharmingen), CD23-PE (Clone EBV CS-5, Biolegend), CD20-BV510 (Clone 2H7, BD Horizon), CD16-PECy7 (Clone 3G8, BD) and CD14-APC (Clone 61D3, Invitrogen). Cell samples were washed once with PBS. Antibodies for cell surface markers and DAPI (final concentration of 2 μg/ml, Sigma-Aldrich) were employed diluted in PBS with 10μg/ml human aggregated IgG, 2% FBS and 2mM EDTA. Cells were incubated with Abs for 15min at 4°C, washed twice with PBS, resuspended in PBS with 2% FBS and 2mM EDTA and analyzed in an LSR II flow cytometer (BD Biosciences). Data were processed with FlowJo X software (TreeStar).

Single-cell suspensions of spleen and LN cells were prepared as previously described [35 (link)]. Joint tissues from hind paws of KSTA mice were dissected free of soft tissue and bones, digested with 100 μg/ml Liberase (Roche) for 45 min at 37^oC, and filtered through a 70-μm-pore cell strainer (SPL Life Sciences) to prepare single cell suspensions of synovial infiltrates. The single cell suspensions were stained with an appropriate combination of mAbs and analyzed by FACS. The mAbs used were: CD138-PE, B220-PerCP or -APC-cy7, FAS-PE, CD19-PerCP or -APC, CD21-FITC or -APC, CD43-APC, CD23-PE-cy7, CD8a-PE, CD25-APC-cy7, CXCR5-biotin, GL7-FITC, CD45.2-FITC, CD27-FITC, Ki-67-FITC, Gr-1-FITC, CD4-FITC or -APC-cy7, phospho-Syk-PE, CD11b-PE or -PerCP, PD-1-APC, CD44-APC-cy7, and F4/80-PerCP mAbs (all from BD Biosciences, eBioscience or Biolegend). Streptavidin-PerCP was purchased from BD Biosciences.

For all staining, cells were stained in PBS with 2% FCS for 20 min at 4°C. BD Aria II was used for flow sorting and BD LSRII was used to collect data. For sorted cells, debris and dead cells were excluded using FSC-SSC. Doublets were excluded using both FSC and SSC singlet gating, then CD19^+ve B cells isolated. For Figures 1C and 2A, CD4 and CD3 stains were also included to exclude contaminating T cells. Antibodies used were specific for and labeled with CD21-FITC, CD3-PE TxR, CD4-PE, IgM-APC, GM-CSF-PE, IL-10-PE, IL-17-alexa fluor647, IFN-γ-FITC (BD Biosciences); CD11b-BV570, CD11b-PE Cy5, CD19-PE, CD19-BV605, CD1d-PerCP Cy5.5, CD21/35-APC, CD23-PE, CD23-alexa fluor 647, CD23-PE Cy7, CD3-FITC, CD3 PE/Dazzle 594, CD38-PE, CD4-PE, CD4-BV605, CD40-PE Cy7, CD43-FITC, CD43-APC, CD5-PE Cy5, CD80-PerCP Cy5.5, CD86-BV421, TIM1-PE, TNFα-BV605 (Biolegend); CD21/35-APC efluor780, CD24-APC efluor780, CD25-efluor450, CD9-APC, DO11.10 TCR-Biotin, IgD-efluor450, MHCII-PE Cy5, CD19-eFluor450, F4/80-APC, IFN-γ-PE Cy7, streptavidin-PE (eBioscience); IgM-TxR, IgM-alexa fluor647 (Southern Biotech); IgM-alexa488, cell tracker green (Molecular Probes), and CFSE (fluka). All analysis was performed using FlowJo Software.

Cells (1× 10⁶) were collected by centrifugation and washed twice in PBS/2% FCS, and the cell pellet was resuspended in 100 μl of PBS/2% FCS. Fluorophore-conjugated surface marker Abs were added at a 1:100 dilution and incubated on ice for 20 min. Cells were washed in PBS/2% FCS and resuspended in PBS/2% FCS for analysis on a BD LSR II Flow Cytometer. The Abs used in the FACS analysis were as follows: CD21-FITC (553818) BD Biosciences Rat, CD23-PE (561773) BD Pharmingen Rat, CD23-Pacific blue (101616) BioLegend Rat, IgD-allophycocyanin (405713) BioLegend Rat, IgM-PE-Cy7 (406513) BioLegend Rat, B220-Pacific blue (558108) BD Biosciences Rat, B220-PE (553089) BD Biosciences Rat, and CD22-PE (BD 553384). The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used to distinguish between live and dead cells. Data were analyzed with FlowJo software. The gating strategy was that cells were initially gated for live lymphocytes followed by analysis of B220⁺ cells.

Single immune cell suspensions were prepared from spleen or peritoneal lavage after red blood cell lysis. Cells were counted on a BD Fortessa Flow cytometer using ‘Fluoresbrite ^TM Calibration Grade 6.0 micron YG microspheres (Polysciences). Cells were incubated with anti-CD16/CD32 (Fc block, clone 2.4G2; BD Bioscience) plus 2% normal mouse and normal rat serum and stained with various combinations of the following Abs in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) for 30 min at 4°C. Murine reactive antibodies, including B220-Pacific Blue, CD3-PerCPCy5.5, CD4-Pacific Blue, CD5-PerCPCy5.5, CD8-APC Cy, CD11b APC Cy7, CD21 FITC, CD23 PE, CD38-FITC, CD95 PE Cy7, CD138-PE, IgD APC Cy7, IgM FITC, GL-7 biotin, Streptavidin BV500, Ki67-APC, Ly6G PE Cy7, and Ly6C PerCP Cy5.5 were all purchased from BD Bioscience.