This application protocol describes the preparation of tetramer solutions, staining, and sorting of SARS-CoV-2–specific B cells using recombinant SARS-CoV-2 spike proteins and the MACSQuant Tyto Cell Sorter.
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 preparation of spike-tetramer solutions, Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin was conjugated to Streptavidin, PE and Strepdavidin, PE-Vio 770 with a molar ratio of 4:1, respectively. For ease of use calculations were performed. Volumetric measurements are shown in table 1.
▲ Note: Always freshly prepare spike-tetramer solutions before the use. Solutions 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 one 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)||MACSQuant Tyto Running 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)||MACSQuant Tyto Running 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 have another isotype. Therefore, the use of REAfinity™ Antibodies for this panel design is not recommended, as REAfinity Antibodies are all IgG1 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 MACSQuant Tyto Running Buffer.
▲Note: Try different volumes of tetramer conjugates in order to achieve the best double staining results (refer to section flow cytometry analysis).
Table 2: 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|
|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.
This protocol is optimized for the activation and expansion of B cells, using the Human CD40-Ligand Multimer Kit (component of the B Cell Expansion Kit, human).
▲Note: Volumes given below are for 10 mL of final expansion medium. When working with larger volumes please scale up accordingly.
▲Note: The final expansion medium should be prepared directly before the stimulation. Long-term storage is not recommended.
For the analysis of spike-specific antibody production of expanded B cell cultures refer to the protocol “Bead-based immunoassay for detection of patient-derived SARS-CoV-2 antibodies”. B cell culture supernatants were processed as described for the plasma or serum samples of COVID-19 convalescents in this protocol. Analysis data is shown in figure 3.
SARS-CoV-2 Spike B Cell Analysis Kit, human (# 130-128-022)
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 (# 130-113-166)
CD27 Antibody, anti-human, Vio Bright FITC (# 130-113-634)
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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