Anti cd19 apc

Cells were suspended with FACS buffer (1 mM EDTA, 0.01% sodium azide, 0.1% bovine serum albumin (BSA), 0.02 M phosphate, 0.15 M NaCl, pH 7.2) and then stained for 20 min with anti-FcR/anti-CD16+CD32/Fc Block (eBioscience) and fluorophore-conjugated antibodies: anti-CD45-PerCP Cy5.5 (BioLegend), anti-Ly6G-AF700 (Thermo Fisher Scientific), anti-CD19-APC (BioLegend), anti-CD11b-PE (BioLegend), anti-NK1.1-PE Cy7 (BioLegend), anti-TCR-beta-Pac Blue (BioLegend), anti-CD4-FITC (BioLegend), and anti-CD8-APC/Cy7 (BioLegend). After staining, cells were washed and then fixed with 1% PFA for 30 min. After fixation, cells were washed three times with 200-μl FACS buffer, and then 50 μl of 123count eBeads Counting beads (Thermo Fisher Scientific) were added to allow for quantification of total number of immune cell subtypes following manufacturer’s instructions. Data were acquired on a LSRII instrument (BD Biosciences). Analysis was performed using FlowJo software, version 10.0.8.

B cells were isolated from PBMCs of convalescent individuals with COVID-19 using immunomagnetic negative selection (EasySep Human B Cell Enrichment Kit, STEMCELL, 17954). In this process, Tetrameric Antibody Complexes and dextran-coated magnetic particles targeted and removed non-B cells. Following isolation, B cells underwent staining with anti-CD19-APC (BioLegend, 302212), anti-IgD-FITC (BioLegend, 348206), anti-IgM–FITC (BioLegend, 314506) phenotyping antibodies, and biotinylated SARS-CoV-2 (BA.4/5) Spike RBD antigen. Viability was assessed using 7-AAD Stain (Invitrogen, 00699342). Class-switched memory B cell-antigen complexes (CD19⁺Ag⁺IgM^-IgD^-7-AAD^-) were then detected with a PE-labeled streptavidin conjugate (BioLegend,127807), and target memory B cells were isolated via flow-cytometric sorting using a BD FACSAira Fusion (BD Biosciences). Subsequently, cells were cultured and activated in human B cell expansion medium for 8 days (ImmunoCultTM Human B Cell Expansion Kit, STEMCELL, 1000645). Flow-cytometric data analysis was conducted using FlowJo version 10.8.1 (Tree Star).

Blood samples and splenocytes were treated with red blood cell lysis buffer and along with lymph node cells and thymocytes were washed with phosphate buffered saline (PBS). They were stained in FACS buffer (PBS, 10% FCS, 0.01% NaN₃) with anti-CD2 FITC, anti-TCRβ PE, anti-CD8 PerCP, anti-CD19 APC and anti-CD4 PB (all Biolegend). Live/Dead discrimination was performed by propidium iodide or Live Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies). Single cells were sorted (BD, MoFlow) onto AmpliGrid slides and processed immediately. For FACS analysis, cells were acquired at BD Canto II and Aria 5. Analysis was performed with FlowJo 9.4 and 10.

Cells were isolated from the spleen and lymph nodes after red blood cell lysis and stained with anti-CD45.1-FITC (BioLegend, 110706), anti-CD45.2-PECy7 (BioLegend, 109830), anti-CD3ε-BV421 (BioLegend, 100227) and anti-CD19-APC (BioLegend, 115512) for 30 min on ice.

Splenocytes were suspended with FACS buffer (1 mM EDTA, 0.01% sodium azide, 0.1% Bovine Serum Albumin (BSA), 0.02 M phosphate, 0.15 M NaCl, pH 7.2) and then stained for 20 min with anti-FcR/anti-CD16 + CD32/Fc Block (eBioscience) and the following fluorophore-conjugated antibodies for NK cell receptor profiling: anti-CD19-APC (BioLegend), anti-CD3-Pac Blue (BioLegend), anti-NK1.1-PE Cy7 (BioLegend), anti-NKG2A-PE (BioLegend), anti-CD11b-PerCP (BioLegend), anti-CD27-AF700 (BioLegend), anti-CX3CR1-BV510 (BioLegend), anti-CD107a-APC Cy7 (BioLegend), and anti-NKG2D-FITC (BioLegend). After staining, cells were washed three times with 200 µl FACS buffer. Data were acquired on a LSRII instrument (BD Biosciences). Analysis was performed using FlowJo software, version 10.0.8.

Mice were infected as detailed in In vivo infections and footpad swelling section, and after 5 days of infection for MAYV or 6 days for CHIKV, the animals were sacrificed, and footpads were removed. Footpads homogenates were obtained after 2 hours incubation in collagenase VIII at 1mg/mL and passed thought a 70 μm cell strainer. Cells were counted and plated in 96-well U bottom. Cells were blocked with Fc Block (BD Biosciences) and then stained for flow cytometry analysis. The antibodies employed were anti-CD3ε-PerCP (BioLegend), anti-CD19-APC (BioLegend), anti-NK1.1-FITC (BioLegend), anti-CD45-APC (BD Biosciences), anti-CD11b-FITC (BioLegend), anti-Ly6G-PerCP (BioLegend) and anti-Ly6C-PE (BioLegend). Cells were also stained with a viability dye (Live/Dead fluorescent dye, Pacific Blue, Life Technologies). Samples were acquired on a FACS Verse flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Peripheral blood mononuclear cells (PBMCs) from SLE patients and HCs were isolated from the heparinized fresh blood by Lymphoprep (Stemcell Technologies, Vancouver, Canada). PBMCs obtained were pre-treated with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with the following fluorescence-conjugated anti-human antibodies: anti-CD3-PE-Cyanine (Cy) 7 (SK7), anti-CD4-Brilliant Violet (BV) 421 (RPA-T4), anti-CD8a-BV510 (RPA-T8), anti-CD19-APC (HIB19), anti-CD56-FITC (HCD56), anti-CD3-PerCP-Cy5.5 (UCHT1), anti-CD19-BV421 (HIB19), anti-CD20-PE-Cy7 (2H7), anti-CD27-BV510 (O323), anti-IgD-FITC (IA6-2), anti-CD38-APC-Cy7 (HIT2; all from BioLegend, San Diego, CA, USA), and anti-CD226-PE (DX11; Miltenyi Biotec). Isotype control antibodies (BD Biosciences, San Jose, CA, USA) were used to determine the level of background staining. Samples were analyzed with the FACS Aria II flow cytometer (BD Biosciences). Data analysis was performed using the FlowJo Software (Tree Star, Ashland, OR, USA). CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), NK cells (CD3⁻CD56⁺), B cells (CD3⁻CD19⁺), naive B cells (CD3⁻CD19⁺IgD⁺CD27⁻), IgD⁺-memory B cells (CD3⁻CD19⁺IgD⁺CD27⁺), switched-memory (SM) B cells (CD3⁻CD19⁺IgD⁻CD27⁺), and plasmablasts (CD3⁻CD19⁺CD20⁻CD38⁺⁺) were all assessed by flow cytometry.