CD38+ plasma B cells were isolated from the PBMC samples by performing two subsequent magnetic separation steps according to the manufacturer’s protocol (Plasma Cell Isolation Kit II, human, Miltenyi Biotec). Briefly, non-plasma B cells are labeled with magnetic beads combined with cocktail antibodies and separated using the MACS column. Then, CD38+ plasma B cells are directly labeled with CD38 MicroBeads and isolated from the pre-enriched B cell pool. Purified CD38+ plasma B cells were eluted and washed in PBS containing 2% (v/v) fetal bovine serum (FBS) and kept for the following RNA isolation step. In order to test the purity of the CD38+ plasma B cells, we also added staining antibodies and 10 μL of Anti-human CD19-BV510 (BioLegend) and CD38-PE-Cy7 (BioLegend) and incubated them for 15 minutes in the dark in the refrigerator (2-8°C). Cells were finally fixed with 4% PFA for 20 minutes on ice. The stained samples were acquired by flow cytometry on a FACS Attune (Invitrogen) and analyzed with FlowJo software (Figure S1 ).
Anti human cd19 bv510
Manufactured by BioLegend
Anti-human CD19-BV510 is a fluorochrome-conjugated antibody that binds to the CD19 cell surface antigen expressed on B cells. It is designed for use in flow cytometry applications to identify and quantify B cell populations.
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4 protocols using anti human cd19 bv510
1
Isolation of CD38+ Plasma B Cells
2
Isolation and Purification of CD38+ Plasma B-cells
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CD38+ plasma B-cells were isolated from the PBMC samples by performing two subsequent magnetic separation steps according to the manufacturer’s protocol (Plasma Cell Isolation Kit II, human, Miltenyi Biotec). Briefly, non-plasma B-cells are labeled with magnetic beads combined with cocktail antibodies and separated using the MACS column. Then, CD38+ plasma B-cell are directly labeled with CD38 MicroBeads and isolated from the pre-enriched B cell pool. Purified CD38+ plasma B-cell were eluted and washed in PBS containing 2% (v/v) fetal bovine serum (FBS) and kept for the following RNA isolation step. In order to test the purity of the CD38+ plasma B cells, we also added staining antibodies and 10 μl of Anti-human CD19-BV510 (BioLegend) and CD38-PE-Cy7 (BioLegend) and incubated them for 15 minutes in the dark in the refrigerator (2–8°C). Cells were finally fixed with 4% PFA for 20 minutes on ice. The stained samples were acquired by flow cytometry on a FACS Attune (Invitrogen) and analyzed with FlowJo software (Fig. S1 ).
3
Isolation and Characterization of SARS-CoV-2 RBD/NTD-Specific B Cells
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B-cells were enriched from the PBMC samples according to the manufacture’s protocol (B Cell Isolation Kit II, human, Miltenyi Biotec). Briefly, non-B-cells are labeled with a cocktail of biotin-conjugated antibodies and separated by the MACS column. Purified B-cells were eluted and kept in the PBS buffer with 2% (v/v) FBS. The enriched B cells were then incubated with 2 μg Biotin-RBD or NTD protein for 30 min at 4°C. After incubation, Anti-Biotin MicroBeads were added and incubated for 30 min. RBD and NTD specific bead binding B cells were washed and eluted in PBS and stored on ice until use. In order to test the purity of the RBD- or NTD-specific B cells, we also added staining antibodies, 10 μl of Anti-human CD19-BV510 (BioLegend), and 2 μg of SARS-CoV-2 RBD-PE or NTD-PE and incubated them for one hour in the dark in the refrigerator (2–8°C). Cells were finally fixed with 4% PFA for 20 minutes on ice. The stained samples were acquired by flow cytometry on a FACS Attune (Invitrogen) and analyzed with FlowJo software (Fig. S1 ).
4
Isolation and Characterization of SARS-CoV-2 Specific B Cells
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B cells were enriched from the PBMC samples according to the manufacture’s protocol (B Cell Isolation Kit II, human, Miltenyi Biotec). Briefly, non-B cells are labeled with a cocktail of biotin-conjugated antibodies and separated by the MACS column. Purified B cells were eluted and kept in the PBS buffer with 2% (v/v) FBS. The enriched B cells were then incubated with 2 μg Biotin-RBD or NTD protein for 30 min at 4°C. After incubation, Anti-Biotin MicroBeads were added and incubated for 30 min. RBD and NTD specific bead binding B cells were washed and eluted in PBS and stored on ice until use. In order to test the purity of the RBD- or NTD-specific B cells, we also added staining antibodies, 10 μL of Anti-human CD19-BV510 (BioLegend), and 2 μg of SARS-CoV-2 RBD-PE or NTD-PE and incubated them for one hour in the dark in the refrigerator (2-8°C). Cells were finally fixed with 4% PFA for 20 minutes on ice. The stained samples were acquired by flow cytometry on a FACS Attune (Invitrogen) and analyzed with FlowJo software (Figure S1 ).
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