This application protocol describes the preparation of tetramer solutions, staining, and flow cytometry analysis of SARS-CoV-2–specific B cells using recombinant SARS-CoV-2 spike proteins.
Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130-091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
The following protocol is an example when using Streptavidin, PE and Streptavidin, PE-Vio 770. Other streptavidin conjugates can be used as well. Always check the concentration of the stock beforehand and adapt the protocol accordingly.
For the preparation of spike-tetramer solutions, the Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin was conjugated to Streptavidin, PE and Strepdavidin, PE-Vio 770 with a ratio of 4 mol to 1 mol, respectively (refer to table 1).
▲ Note: Always prepare spike-tetramer solutions fresh before the use. They can be stored for up to 24 hours at 2–8 °C, protected from light.
Each test indicated in table 1 corresponds to 5×106 B cells (or 107 PBMCs). When working with less than 5×106 B cells (or 107 PBMCs), use the same volumes as indicated for 1 test. When working with more than 5×106 B cells (or 107 PBMCs), scale up all reagent volumes accordingly.
Table 1: Volumes of spike-tetramer solutions
|No. of tests||Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin (0.1 mg/mL)||Streptavidin, PE (50 µg/mL)||PEB buffer|
|1||3 µL||0.6 µL||1.4 µL|
|5||15 µL||3 µL||7 µL|
|10||30 µL||6 µL||14 µL|
|20||60 µL||12 µL||28 µL|
|No. of tests||Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin (0.1 mg/mL)||Streptavidin, PE-Vio 770 (50 µg/mL)||PEB buffer|
|1||6 µL||1.2 µL||2.8 µL|
|5||30 µL||6 µL||14 µL|
|10||60 µL||12 µL||28 µL|
|20||120 µL||24 µL||56 µL|
▲ Note: If using an IgG Antibody, the other antibodies should not be of IgG isotype. Therefore, the use of REAfinity™ Antibodies for this panel design is not recommended, as REAfinity Antibodies are all IgG isotype.
For each test (5×106 B cells or 107 PBMCs) mix 2 μL of each fluorochrome-conjugated antibody, 5 μL of 7-AAD Staining Solution, 5 μL of spike‑tetramer-PE, and 10 μL of spike-tetramer-PE-Vio 770.
Fill up to 100 μL with PEB buffer.
▲Note: Try different volumes of tetramers conjugates in order to achieve the best double staining results (refer to section Flow cytometric analysis).
Suggested antibody panel
|V1||IgG Antibody, anti-human, VioBlue®||IS11-3B2.2.3|
|V2||IgA Antibody, anti-human, VioGreen™||IS11-8E10|
|B1||CD27 Antibody, anti-human, Vio Bright FITC||M-T271|
|B3||7-AAD Staining Solution||7-AAD|
|R1||IgM Antibody, anti-human, APC||PJ2-22H3|
|R2||CD19 Antibody, anti-human, APC-Vio 770||LT19|
B cells can be enriched from single-cell suspension of peripheral blood mononuclear cells (PBMCs) using the REAlease® CD19 MicroBead Kit (# 130-117-034) accordingly to the data sheet including removal of magnetic labeling.
Antigen-specific B cells are relatively rare cells. The detection of such cells requires the acquisition of a large number of events. Pre-enrichment of B cells using, e.g., the REAlease® CD19 MicroBead Kit (# 130-117-034), prior to flow cytometry will decrease the total number of cells which need to be acquired in order to detect rare events.
PEB buffer: phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA
MACSQuant® Analyzer 10 (# 130-096-343) or other flow cytometers equipped with violet (405 nm) and red (635 nm) lasers able to discriminate FITC, PE, and APC fluorescence.
MACS Chill 96 Rack (# 130-094-459) when using MACSQuant Analyzer 10
MACSQuant Calibration Beads (# 130-093-607) when using MACSQuant Analyzer 10
MACS Comp Bead Kits for optimal compensation of the fluorescence spillover from fluorochrome-conjugated antibodies
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