PBMCs were stained with SARS-CoV-2 Spike and RBD probes, as previously described (Juno et al., 2020 (link)). Probes were generated by sequential addition of streptavidin-phycoerythrin (PE) (Thermo Fisher Scientific) to trimeric S protein biotinylated using recombinant Bir-A (Avidity), while SARS-CoV-2 RBD was labeled to APC using an APC Conjugation Lightning-Link Kit (Abcam). PBMCs were surface stained with Aqua viability dye (Thermo Fisher) and monoclonal antibodies against CD19-ECD (#IM2708U, Beckman Coulter), IgM BUV395 (#563903), CD21 BUV737 (#564437), IgD PE-Cy7 (#561314), IgG BV786 (#564230), streptavidin-BV510 (#563261) (BD Biosciences), CD20 APC-Cy7 (#302314), CD14 BV510 (#301841), CD3 BV510 (#317332), CD8a BV510 (#301048), CD16 BV510 (#302048), CD10 BV510 (#312220) and CD27 BV605 (#302829) (BioLegend). Cells were washed, fixed with 1% formaldehyde and acquired on a BD LSRII Fortessa.
Igg bv786
Manufactured by BD
IgG BV786 is a fluorescent-labeled antibody used for flow cytometry analysis. It is designed to detect immunoglobulin G (IgG) in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd14 bv510, by BioLegend (1 mentions)
Cd16 bv 510, by BioLegend (1 mentions)
Lsrii fortessa, by BD (1 mentions)
Cd3 bv510, by BioLegend (1 mentions)
Cd27 bv605, by BioLegend (1 mentions)
Cd10 bv510, by BioLegend (1 mentions)
Cd8a bv510, by BioLegend (1 mentions)
Cd20 apc cy7, by BD (1 mentions)
Aqua viability dye, by Thermo Fisher Scientific (1 mentions)
Streptavidin phycoerythrin pe, by Thermo Fisher Scientific (1 mentions)
Recombinant bir a, by Avidity (1 mentions)
Lightning link apc conjugation kit, by Abcam (1 mentions)
Cd21 buv737, by BD (1 mentions)
Streptavidin bv510, by BD (1 mentions)
Cd19 ecd, by Beckman Coulter (1 mentions)
Igd pe cy7, by BD (1 mentions)
Cd56 fitc, by BD (1 mentions)
Cd14 fitc, by BD (1 mentions)
Live dead fixable aqua dye, by Thermo Fisher Scientific (1 mentions)
Cd16 fitc, by BD (1 mentions)
3 protocols using igg bv786
1
SARS-CoV-2 Spike and RBD Staining of PBMCs
2
SARS-CoV-2 Spike-Specific Single B Cell Sorting
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BA.4/5 S-specific or BA.4+all S-specific single B cell sorting was performed as previously described7 (link),26 (link). Briefly, PBMC were stained with LIVE/DEAD Fixable Aqua dye (Invitrogen) followed by recombinant trimeric S-twin-Strep of BA.4/5 or BA.4+all. Cells were then incubated with CD3-FITC (1:10 dilution, BD, Cat. 555332), CD14-FITC (1:10 dilution, BD, Cat 555397), CD16-FITC (1:10 dilution, BD, 555406), CD56-FITC (1:50 dilution, BD, Cat. 562793), IgM-FITC (1:10 dilution, BD, Cat. 555782), IgA-FITC (1:50 dilution, Dako, Cat. F0188), IgD-FITC (1:10 dilution, Dako, Cat. F0189), IgG-BV786 (1:20 dilution, BD, Cat. 564230) and CD19-BUV395 (1:50 dilution, BD, Cat. 563549), along with Strep-MABS-DY549 (1:50 dilution, iba, Cat. 2-1566-050) to stain the twin strep tag of the S protein. IgG+ memory B cells were gated as CD19+, IgG+, CD3−, CD14−, CD56−, CD16−, IgM−, IgA− and IgD−, and S+ were further selected, and single cells were sorted into 96-well PCR plates with 10 µl of catching buffer (Tris, Nuclease free-H2O and RNase inhibitor). Plates were briefly centrifuged at 2000×g for 1 min and left on dry ice before being stored at −80 °C.
3
Single-Cell Sorting of HIV Env-Specific B Cells
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Cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed and transferred to warm thaw media supplemented with benzonase [RPMI, 10% fetal bovine serum (FBS), 1% L-Glutamine, 1% penicillin-streptomycin, benzonase 50 U/mL] followed by two additional washes with thaw media. Samples were rested overnight at 37°C 5%
CO2 and centrifuged at 250 x g for 10 minutes before transferring to a 96-well U-bottom plate for staining. Cells were pelleted for 3 minutes at 750 x g and resuspended in Env gp120 positive and HIV-11086 Env gp120 negative) were bulk sorted into separate FACS tubes containing lysis buffer [DNA suspension buffer (Teknova), 10% BSA, 2.5% RNAseOut] using the following gating strategy: singlets, lymphocytes, Live/Dead-, CD3-, CD56-, CD14-, CD19+ IgD-, IgG+, 1086 gp120+ or 1086 gp120-.
Four of the original subset were selected for further single-cell analysis and were thawed and stained as described above with the following modifications: IgD BV650 (BD Biosciences, IA6-2) and IgG BV786 (BD Biosciences, G18-145). Cells that were 1086 gp120+ were single cell sorted using the gating scheme described above into PCR plates containing lysis buffer. Plates with sorted cells were sealed and frozen on dry ice prior to transferring to -80°C for storage.
CO2 and centrifuged at 250 x g for 10 minutes before transferring to a 96-well U-bottom plate for staining. Cells were pelleted for 3 minutes at 750 x g and resuspended in Env gp120 positive and HIV-11086 Env gp120 negative) were bulk sorted into separate FACS tubes containing lysis buffer [DNA suspension buffer (Teknova), 10% BSA, 2.5% RNAseOut] using the following gating strategy: singlets, lymphocytes, Live/Dead-, CD3-, CD56-, CD14-, CD19+ IgD-, IgG+, 1086 gp120+ or 1086 gp120-.
Four of the original subset were selected for further single-cell analysis and were thawed and stained as described above with the following modifications: IgD BV650 (BD Biosciences, IA6-2) and IgG BV786 (BD Biosciences, G18-145). Cells that were 1086 gp120+ were single cell sorted using the gating scheme described above into PCR plates containing lysis buffer. Plates with sorted cells were sealed and frozen on dry ice prior to transferring to -80°C for storage.
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