Microchip-based cell sorting and expansion of SARS-CoV-2–specific B cells using recombinant SARS-CoV-2 proteins and the MACSQuant® Tyto® Cell Sorter

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.

Protocol

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).

Preparation of spike-tetramer solutions

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.

  1. Resuspend the Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin in deionized, sterile-filtered water to a final concentration of 0.1 mg/mL (e.g. use 250 µL water for 25 µg protein). 
    Note: To avoid repeated freeze-thaw cycles prepare working aliquots from the stock solution of the Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin. Store the working aliquots at –70 °C. Once thawed the protein can be stored for up to three days at 2–8 °C.
  2. Dilute Streptavidin, PE as well as Streptavidin, PE-Vio 770 to a concentration of 50 µg/mL in MACSQuant Tyto Running Buffer. 
    Note: Check the concentration of the streptavidin conjugates and dilute to 50 µg/mL, if needed.
  3. Mix Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin, Streptavidin, PE or Streptavidin, PE-Vio 770, and MACSQuant Tyto Running Buffer carefully as indicated in table 1 and incubate for 15 minutes at room temperature.

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 testsRecombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin (0.1 mg/mL) Streptavidin, PE (50 µg/mL)MACSQuant Tyto Running Buffer
3 µL0.6 µL1.4 µL
15 µL 3 µL 7 µL 
10 30 µL 6 µL 14 µL 
20 60 µL12 µL 28 µL 
Spike-tetramer-PE-Vio 770  
No. of testsRecombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin (0.1 mg/mL) Streptavidin, PE-Vio 770 (50 µg/mL) MACSQuant Tyto Running Buffer
16 µL1.2 µL2.8 µL
530 µL 6 µL 14 µL 
1060 µL 12 µL 28 µL 
20120 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.

Preparation of the antibody staining mix

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 the MACSQuant Tyto Running 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​FluorochromeClone
V1IgG Antibody, anti-human, VioBlue®IS11-3B2.2.3
V2IgA Antibody, anti-human, VioGreen™IS11-8E10
B1CD27 Antibody, anti-human, Vio Bright FITCM-T271
B2Spike-tetramer-PE 
B37-AAD Staining Solution 
R1IgM Antibody, anti-human, APCPJ2-22H3
R2CD19 Antibody, anti-human, APC-Vio 770LT19
B4Spike-tetramer-PE-Vio 770 

(Optional) Enrichment of CD19+ cells 

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.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.
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