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2 protocols using anti igd bv 605

1

Comprehensive Murine Immune Cell Analysis

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Anti-CD16/CD32 (BD, 2.4G2 [anti-FcγR]) was used as a blocking reagent and PI-PECF594 (Invitrogen, CAT P3566) was used to identify dead cells. The following antibodies and reagents were used for germinal center B-cell staining: anti-IgD-BV-605 (BioLegend,11-26c.2a), anti-B220-APCeFlour780 (eBioscience, RA3-6B2), anti-CD38-Alexa 700 (eBioscience, 90), and anti-CD95-PE-CY7 (BD, Jo2). For T follicular helper cell (Tfh) staining, anti-CXCR5 (BD, 2G8), anti-rat IgG (fab’) Alexa-647 (Jackson Immuno Research, Code:712-606-153). 2W-specific T-cells were identified using a 2W:I-Ab tetramer labeled with PE (50959 I-A(b)) or BV421(50960 I-A(b) (NIH Tetramer Core Facility), and the following antibodies: anti-CD3-Alexa flour 700 (BioLegend, 17A2), anti-CD4-FITC (BioLegend, RM4-5), anti-B220-APCeFlour780 (BioLegend, RA3-6B2), and anti-PD1-PE-CY7 (eBioscience, J43). The Click-iT™ Plus EdU Alexa fluor 647 Flow cytometry kit was used for EdU staining of newly synthesized DNA (Invitrogen, CAT C10634). Porcine cardiac myosin (Sigma-Aldrich, CAT: M0531) and a cardiac myosin monoclonal antibody (ThermoFisher, CAT: MA1-26180) was used for investigation of myosin-reactive antibody responses.
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2

Characterization of SARS-CoV-2 antibody responses

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Cryopreserved PBMCs were centrifuged and suspended in PEB buffer (PBS with 0.5% BSA and 2 mM EDTA) and incubated with Fc receptor block (Miltenyi, 130-059-901) for 15 min at 4 °C (dilution 1:10). Next, cells were washed in PEB and stained for 30 min in brilliant stain buffer at 4 °C in the dark using the following antibodies together with both the PE-and APC-conjugated recombinant RBD tetramers: anti-CD3-APC Fire 810 (BioLegend, 344858; diluted 1:100); anti-CD11c-BV785 (Bio-Legend, 301644; 1:50); anti-CD19-PE Vio770 (Miltenyi, 130-113-170; 1:100); anti-CD20-BV421 (BD, 562873; 1:100); anti-CD21-BUV496 (BD, 750614; 1:50); anti-CD27-PercP-Vio700 (Miltenyi, 130-113-632; 1:100); anti-CD38-Viobright FITC (Miltenyi, 130-113-433: 1:50); anti-IgM-PE-CF594 (BD, 562539; 1:50); anti-IgD-BV605 (BioLegend, 348232; 1:50); and anti-IgG-BV480 (BD, 746341; 1:50). Cells were washed in PEB and resuspended in a PEB dilution (1:500) of the fixable viability dye eFluor 780 (eBiosciences, 65-0865-18). They were next washed and fixed with 4% paraformaldehyde for 20 min at 4 °C in the dark before a final wash and resuspension for analysis. Cells were then acquired on a Cytek Aurora spectral flow cytometer equipped with five lasers operating at 355 nm, 405 nm, 488 nm, 561 nm and 640 nm using the Spectro-Flo V.2.2.0 (Cytek) software. Data were analysed using FlowJo 10.6.1 software (BD).
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