Anti cd45r pe

Cells from blood, spleen, and lymph nodes were incubated with 1 µL per sample of mouse anti-Rat CD32 (clone D34-485, 20 µg/mL, BD biosciences, San Jose, CA, USA) diluted in staining buffer (0.5% bovine serum albumin (BSA) and 0.01% sodium azide in PBS), to prevent unspecific bindings. Then, cells were immunostained for 30 min on ice for the presence of T cells, B cells, NK cells, and myeloid cells by the following surface antibodies: anti-TCR-PerCP (clone R73, 4 µg/mL), anti-CD4-APC (clone OX-35, 4 µg/mL), anti-CD8a-V450 (clone OX-8, 8 µg/mL), anti-CD45R-PE (clone His24, 8 µg/mL), anti-CD161a-FITC (clone10/78, 2 µg/mL), anti-CD11b/c-PE-Cy7 (clone OX-42, 1.6 µg/mL), anti-CD40-FITC (clone HM40-3, 40 µg/mL), anti-CD86-PE (clone 24F, 16 µg/mL) all from BD biosciences. Cells stained with the corresponding isotype antibodies were used as control. Cells were washed 4 times in PBS and fixed with paraformaldehyde 1%. Samples were analyzed on a FACSCanto flow cytometer (Becton Dickinson), with acquisition of 10.000 events in gate for each sample, and data analyzed with FlowJo software.

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).

Immune cells were isolated and flow cytometry was performed as previously described [9 (link),10 (link)]. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood over Histopaque-1083 (Cat# 10831, Sigma-Aldrich, Saint Louis, MO, USA). Approximately 1 × 10 ⁶ cells were stained with an antibody cocktail containing anti-CD45 PE-Cy7 (BioLegend, catalog no. 202214, clone OX-1, 0.062 µg, San Jose, CA, USA), or anti-CD3 PerCP eFluor710 (eBioscience, catalog no. 46-0030-82, clone G4.18, 0.062 µg), or anti-CD8a FITC (BioLegend, catalog no. 201703, clone OX-8, 0.125 µg), or anti-CD4 APC-Cy7 (BioLegend, catalog no. 201518, clone W3/25, 0.062 µg), or anti-CD11b/c Alexa eFluor 660 (eBioscience, catalog no. 50-0110-82, clone OX-42, 0.062 µg), or anti-CD45R PE (BD Bioscience, catalog no. 554881, clone HIS24, 0.125 µg) in 100 µL of wash buffer [PBS (1×) without Ca²⁺ and Mg²⁺, 2% FBS, and 2 mM EDTA]. Excess antibody was washed out and cells then resuspended in 300 µL of wash buffer. DAPI was added for quantification of dead cells 10 min before samples were run on an LSR II flow cytometer (BD Biosciences, San Jose, CA, USA) acquired and analyzed with FlowJo Software (FlowJo, Ashland, OR, USA).