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As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is committed to providing our customers around the world with the highest quality products. In addition to direct selling in more than 20 countries in North America, Europe and Asia/Pacific, Miltenyi Biotec also provides support for our customers through an extensive distributor network covering dozens of additional countries.
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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.
Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin, Streptavidin, PE, Streptavidin, PE-Vio® 770, and fluorochrome-conjugated antibodies as well as the 7-AAD Staining Solution are available as single products or all-in-one in the SARS-CoV-2 Spike B Cell Analysis Kit, human (# 130-128-022).
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
Spike-tetramer-PE | |||
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 |
Spike-tetramer-PE-Vio 770 | |||
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 IgG as 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 cytometry analysis).
Suggested antibody panel
Channel | Fluorochrome | Clone |
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 |
B2 | Spike-tetramer-PE | |
B3 | 7-AAD Staining Solution | 7-AAD |
R1 | IgM Antibody, anti-human, APC | PJ2-22H3 |
R2 | CD19 Antibody, anti-human, APC-Vio 770 | LT19 |
B4 | Spike-tetramer-PE-Vio 770 |
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
SARS-CoV-2 Spike B Cell Analysis Kit, human (# 130-128-022)
or
Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin (# 130-127-682)
Streptavidin, PE (# 130-106-789)
Strepdavidin, PE-Vio® 770 (# 130-106-793)
Deionized, sterile-filtered water
SARS-CoV-2 Spike B Cell Analysis Kit, human (# 130-128-022)
or
7-AAD Staining Solution (# 130-111-568)
IgA Antibody, anti-human, VioGreen™ (# 130-113-481)
IgG Antibody, anti-human, VioBlue® (# 130-119-881)
IgM Antibody, anti-human, APC (# 130-122-915)
CD19 Antibody, anti-human, APC-Vio 770 (# 130-113-165)
CD27 Antibody, anti-human, Vio Bright FITC (# 130-113-634)
REAlease® CD19 MicroBead Kit (# 130-117-034)
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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