Enrichment of SARS-CoV-2–specific B cells using recombinant SARS-CoV-2 proteins

Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin, Streptavidin, PE, and Anti-PE MicroBeads UltraPure are available as single products or all-in-one in the SARS-CoV-2 Spike B Cell MicroBead Kit, human (# 130-128-031).

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. 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 the spike-tetramer-PE solution, the Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin was conjugated to Streptavidin, PE (refer to table 1).

▲ Note: Always prepare the spike-tetramer solution 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 4 °C.
2. Dilute Streptavidin, PE to a concentration of 50 µg/mL in PEB buffer.
3. Mix Recombinant SARS-CoV-2 Spike-Prot (HEK)-Biotin, Streptavidin, PE, and PEB 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.

B cells must 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 the REAlease Complex.

▲ CD19⁺ cells were enriched from single-cell suspension of peripheral blood mononuclear cells (PBMCs) of COVID-19 convalescent individuals using the REAlease CD19 MicroBead Kit. All data show CD19⁺ cells after freezing and thawing.

▲ Volumes for magnetic labeling given below are for up to 5×10⁶ total B cells. When working with fewer than 5×10⁶ B cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 10⁷ total B cells, use twice the volume of all indicated reagent volumes and total volumes).

1. Determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend cell pellet in 100 µL of PEB buffer per 5×10⁶ total B cells. Add 10 µL of spike-tetramer-PE and incubate for 30 minutes at 2–8 °C. Aspirate supernatant completely.
4. Wash cells by adding 1–2 mL of PEB buffer per 10⁶ total B cells and centrifuge at 300xg for 10 minutes. Aspirate supernatant completely. 
5. Resuspend cell pellet in 160 µL of PEB buffer per 10⁶ total B cells.
6. Add 40 µL of Anti-PE MicroBeads UltraPure (# 130-105-639) per 10⁶ total B cells.
7. Mix well and incubate for 15 minutes in the refrigerator (2–8 °C).
8. Wash cells by adding 1−2 mL of buffer per 10⁶ total B cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely. 
9. Resuspend up to 10⁷ cells in 500 μL of buffer.
   ▲ Note: For higher cell numbers, scale up buffer volume accordingly.
10. Proceed to magnetic separation.

▲ Always wait until the column reservoir is empty before proceeding to the next step.

Magnetic separation with MS Columns (# 130-042-201)

1. Place column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column data sheet.

2. Prepare column by rinsing with 500 μL of buffer.

3. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells.

4. Wash column with 3×500 μL of buffer. Collect unlabeled cells that pass through and combine with the flow-through from step 3.

▲ Note: Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty.

5. Remove column from the separator and place it on a suitable collection tube.

6. Pipette 1 mL of buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.