Anti gl7 fitc

Single cell suspensions were stained using combinations of anti-CD11c-PE (N418, BD Biosciences, San Jose, CA, USA), anti-B220-PerCP (RA3-6B2, BD Biosciences), anti-I-A^b/Ea_52-68-biotin (YAe, eBioscience, San Diego, CA, USA), anti-CD4-eFluor450 (RM4-5, eBioscience), anti-ICOS-alexa-fluor-488 (C398.4A, BioLegend, San Diego, CA, USA), anti-PD-1-PE-Cy7 (J43, eBiosciences), anti-SLAM-APC (TC15-12F12.2, BioLegend), biotinylated anti-CXCR5 (2G8, BD Biosciences), anti-B220-eFluor450 (RA3-6B2, eBioscience), anti-GL-7-FITC (BD Biosciences), anti-FAS-PE (BD Biosciences) and biotinylated anti-IgM^a (DM-1, BD Biosciences). Biotinylated antibodies were detected by incubation with fluorochrome-conjugated streptavidin (BD Biosciences). Appropriate isotype controls were used throughout. Samples were acquired using a MACSQuant analyzer (Miltenyi Biotec, Surrey, UK) and analysed using FlowJo software (Tree Star Inc, Ashland, OR, USA).

C57BL/6 (WT) mice were purchased from The Jackson Laboratories (Bar Harbor, ME).
Cd244^-/- mice on the C57BL/6 background [12 (link)], were kindly provided by Dr. Raymond Welsh (University of Massachusetts Medical Center, Worcester, MA). Mice were maintained in the Translational Biomedical Research Center of the Medical College of Wisconsin and both genders were used for experiments in an age (6–12 week) and gender-matched manner. anti-CD45R-PE-Texas Red, anti-CD45R-PE, anti-GL7-FITC and anti-CD5-APC were purchased from BD Biosciences (San Jose, CA). CD21-eFluor 450, anti-CD23-PE-Cy7, anti-CD93-biotin, anti-CD4-APC-eFluor 780, anti-CD8-PE-Cy7, anti-TCRβ-FITC, anti-TCRβ-PE, anti-CD11b-Alexa Fluor 488, anti-CD244, anti-Foxp3-PE, anti-CD19-Alexa 700, anti-CD4-PE, anti-IgG-FITC and Streptavidin PE-Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-CD11b-Brilliant Violet 605, anti-CD11c-PE, anti-NK1.1-APC, anti-IgM-APC Cy7, anti-IgD-Pacific Blue, anti-CD38-Alexa Fluor 647, anti-CD138-APC and rat anti-mouse IgG_2a-biotin were purchased from Biolegend (San Diego, CA). Anti-IgM-FITC and the SBA Clonotyping System-B6/C57J-HRP were purchased from Southern Biotech (Birmingham, AL). 4-Hydroxy-3-nitrophenylacetyl (NP)-ficoll, NP-Chicken Gamma Globulin (CGG), NP(24)-PE and NP-BSA were purchased from Biosearch Technologies (Novato, CA).

The frequency of T_FH and GC B cells from draining ILNs was assessed by flow cytometry. For T_FH, 1 × 10⁶ cells were stained with anti-CD3 (PerCP-Cy5.5), anti-CD4 (V500), anti-CD8 (APC-Cy7), anti-CD19 (Alexa 700), anti-CXCR5 (biotin), streptavidin (APC), anti-PD-1 (PE), and anti-Bcl-6 (V450) (BD Biosciences, San Diego, CA, USA). Intracellular staining for Bcl-6 was performed with a Foxp3 staining kit (eBioscience, Carlsbad, CA, USA) according to the manufacturer’s instructions. Data was expressed as the frequency (%) of CD4+ T cells expressing CXCR5 + PD-1 + BCL-6+ markers. For GC B cells, 1 × 10⁶ cells were stained with anti-CD3 (PerCP-Cy5.5), anti-CD4 (V500), anti-CD19 (Alexa 700), anti-CD95 (PE-Cy7), and anti-GL-7 (FITC) (BD Biosciences). Data was expressed as the frequency (%) of B cells expressing CD95 + GL-7+ markers. A total of 200,000 events were collected and analyzed by flow cytometry (LSR Fortessa, BD Biosciences, San Diego, CA, USA) using the FACS Diva (BD Bioscience, San Diego, CA, USA) and FlowJo 10.0.6 (BD Bioscience, San Diego, CA, USA) software programs. Fluorescence Minus One (FMO) controls were performed to confirm proper compensation and define positive signals.

To quantify germinal center (GC) B cells and follicular helper T-cells (Tfh) we stained isolated draining lymph node cells with anti-CD4 pacific blue, anti-B220 PercP, anti-PD-1 PE, anti-CXCR5 biotin, anti-CD95 PE, anti-GL7 FITC and streptavidin APC (all from BD Biosciences). One million events in a live lymphocyte gate were acquired on a FACSCanto flow cytometer (BD Biosciences) and then analyzed using FlowJo software (version 10, Tree Star). Germinal center B cells were identified as CD4^-B220⁺GL7⁺CD95⁺ and Tfh as CD4⁺B220^-PD1⁺CXCR5⁺ population. S1 Fig shows the gating strategy used to identify each cell population.

Kidneys and spleens of NZB/W mice were embedded in OCT compound (Sakura Finetek, Torrance, CA, USA) and snap-frozen in liquid nitrogen. Frozen sections were fixed in acetone and blocked with 10% normal donkey serum (Sigma Aldrich). Kidney sections were stained with a 1:200 dilution of anti-IgG-biotin (Sigma Aldrich), anti-IgM-biotin (Southern Biotech) and anti-C3-biotin (Bioss), followed by reaction with 1:200-dilution of streptavidin-cy3 (Invitrogen). Spleen sections were stained with anti-GL7-FITC, anti-CD4-APC, and anti-IgD-PE (all from BD Biosciences or eBioscience). Fluorescence images were acquired using a TCS SP5 confocal microscope (Leica). The area of GCs composed with GL7⁺ cells per image was calculated using ImageJ software (NIH, Bethesda, MD, USA).

Suspensions of single lymphocytes from spleens and Payer’s patches were prepared and stained with antibodies as follows: anti-CD3-pacific blue, anti-CD19-PE/Cy7, anti-CD38-APC, anti-CD138-biotin, anti-Fas-PE/CF594, anti-GL7-FITC and Streptavidin-PE from BD Biosciences (California, USA). The lymphocytes from bone marrows were isolated and stained with anti-CD3-pacific blue, anti-CD19-PE/Cy7, and anti-CD138-biotin. Flow cytometry data were acquired by the flow cytometer (CantoII; BD Biosciences) and analyzed with FlowJo software (version 10; Tree Star Inc, Oregon, USA).

Mouse kidneys and spleens were assayed post mortem by fluorescence immunohistochemical methods as described^{27 (link)}. Frozen sections were stained with appropriate combinations of anti-B220-allophycocyanin (eBioscience), anti-GL7-FITC (BD Biosciences), anti-IgG-biotin (Sigma-Aldrich), and anti-IgM-biotin (Southern Biotech) Abs and streptavidin. GCs were counted at a magnification of X200, and glomerular Ig deposits were scored as mean fluorescence intensities using ImageJ software (NIH).