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

1. Resuspend up to 5x10⁶ B cells in 100 μL of antibody staining cocktail. Incubate for 30 minutes at 2–8 °C.
2. Wash cells by adding 1–2 mL of MACSQuant Tyto Running Buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend cell pellet in a suitable amount of MACSQuant Tyto Running Buffer and proceed to sorting.
   ▲Note: Resuspend cautiously as B cells are very sensitive. Shear forces induce stress, what could lead to lower viability of the cells and unspecific staining.
   ▲Note: The MACSQuant Tyto Cell Sorter can sort between 100 µL and 10 mL. It is recommended to use 2–10 mL volumes for optimal performance.

• B cells have been pre-enriched using the REAlease CD19 MicroBead Kit (# 130-117-034).
• Refer to the MACSQuant Tyto Cell Sorter user manual and MACSQuant Tyto Cartridge data sheet for instructions on how to use the instrument and cartridges.

1. Load B cells into the input chamber of a MACSQuant Tyto Cartridge according to the MACSQuant Tyto Cartridge data sheet.
   ▲Note: Take an aliquot of the original fraction from the input chamber for further cell analysis after sorting.
2. Start the MACSQuant Tyto Cell Sorter and software.
3. Load a workspace (gating strategy is shown in figure 1).
   ▲Note: Compensate panel if needed.
4. (Optional) Adjust sample volume in the Experiment tab according to the particular sample.
5. Load the MACSQuant Tyto Cartridge into the MACSQuant Tyto Cell Sorter following the instructions in the cartridge data sheet.
6. Click the Start measurement button in the status bar to initialize data acquisition.
7. Adjust PMTs, trigger, and gating settings.
   ▲Note: Adjust compensation if needed.
8. Click the Sort button in the status bar to start the sort process.
9. Click the Stop measurement button in the status bar and take out the cartridge.
   ▲Note: After the input chamber is empty, the sort will automatically stop.
10. Collect the sorted cells from the positive collection chamber and the non-target cells from the negative collection chamber. 
    ▲Note: To increase the purity of target cells, the sorted cells can be sorted again.
11. Take an aliquot from both fractions and proceed to flow cytometry analysis using a MACSQuant Analyzer 10.
    ▲Note: Sorted B cells are already stained and just need to be applied to the flow cytometer for analysis.

1. Reconstitute lyophilized Human CD40-Ligand (component of the B Cell Expansion Kit, human) with deionized sterile-filtered water to a final concentration of 0.5 mg/mL in a volume of 200 μL (for 100 μg size) or 1000 μL (for 500 μg size) and store at –20 °C. 
2. Reconstitute human IL-4 with deionized sterile-filtered water to a final concentration of 2.5×10⁵ U/mL, e.g., if the lot has a biological activity of 8×10⁶ U/mg, 160 μL deionized sterile-filtered water should be used for reconstitution of 5 μg.
3. Mix components by pipetting up and down until resuspended. To avoid repeated freeze-thaw cycles, reconstituted reagents should be aliquoted and stored at –20 °C or below until use.

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. 

1. To increase the biological activity, Human CD40-Ligand and Cross-Linking Antibody (component of the B Cell Expansion Kit, human) must be pre-incubated. Mix 40 μL reconstituted Human CD40-Ligand with 40 μL Cross-Linking Antibody. Incubate for 30 minutes at room temperature (19–25 °C).
2. Prepare 10 mL StemMACS™ HSC Expansion Media XF (component of the B Cell Expansion Kit, human) with 2 µL reconstituted Human IL-4, and 5% AB serum. Add the multimerized Human CD40-Ligand (80 μL) to prepared media to get the final expansion medium.
3. Remove cells from the sort chamber using HSC Expansion Media with all additives in three sequential steps using 200 µL per step to wash out the sorting chamber. Count cells to determine the total cell number.
4. Spin cells down at 200×g for 10 min and resuspend them in appropriate amount of Expansion medium with all indicated additives. Plate at least 3000 cells/mL and up to 7500 cells/mL in 200 µL of the final expansion medium in a 96-U bottom cell culture plate. The same number of cells should be plated for all control fractions within one experiment.
   ▲Note: For higher cell numbers plate cells as indicated in the data sheet of the B cell Expansion Kit.
5. Incubate at 37 °C, 5% CO₂.
6. On days 7 and 10 exchange half of the medium with fresh medium supplemented with 5% AB serum and 50 units IL-4/mL (0.2 μL human IL-4 per mL medium from step 2). Harvested supernatants can be stored at –20°C and used for downstream applications, i.e., tested for the presence of spike-specific Ig.
   ▲Note: Pipette gently to avoid cell loss as B cell grow in non-adherend cell clusters.
7. On day 14 harvest B cells by pipetting gently and optionally use them for further downstream applications. 
   ▲Note: Expansion rates calculated from cell counts are shown in figure 3.

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.