This application protocol describes the preparation of tetramer solutions, the labeling, and the enrichment of SARS-CoV-2–specific B cells using recombinant SARS-CoV-2 spike 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.
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-PE solution
|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|
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).
▲ 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.
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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