Sorting of antigen-specific single B cells by flow cytometry

please receive attached protocol


Sorting of antigen-specific single B cells by flow cytometry

The method of antigen-specific single B cell sorting was described in a previous study [19]. In the current study, 80-200 bp dsDNAs from calf thymus were purified by gel electrophoresis and biotinylated according to the manufacturer’s protocol (VECTOR, Burlingame, CA). Biotinylated dsDNA was confirmed by ELISA. 50 μL aqua blue was used to exclude dead cells in about 25 million PBMCs on D14 post-vaccination. Next, 50 μL antibody cocktail containing anti-CD3-PerCP (OKT3), anti-CD14-APC-cy7 (HCD14), anti-CD19-BV421 (HIB19), anti-CD20-PE-cy7 (L27), anti-IgM-APC (G20-127), anti-IgG-PE (G18-145), and 15 μL biotinylated dsDNA (130 ng/μL) was surface stained for 1 h at 4℃. To sort HA-specific single B cells, 10 μL biotinylated HA (200 ng/μL, Emory University) was used. After 3 times washings with PBS, 50 μL of 1:1000 diluted streptavidin-FITC (BD Pharmingen) was added and incubated for 15 min at 4℃. Antigen-specific IgG+ single B cells (CD3–CD14–CD19+CD20+IgG+ dsDNA+ or HA+) were identified and sorted into a 96-well PCR plate containing 10μL of catching buffer per well using FACS Aria II cell sorter (BD Biosciences, San Jose, CA). The catching buffer contained 2 μL of 10 x first strand buffer (Invitrogen, Carlsbad, CA) 2 μL of 0.1 M DTT (Invitrogen), 1 μL of RNase Out (Invitrogen), and 0.05 μl of igepal (Sigma-Aldrich, St. Louis, MO). The sorted cells were stored at –80℃ for future uses, flow cytometry data was analyzed by FlowJo software (Version 10.0.8).

Single B cell immunoglobulin gene amplification and antibody expression 

As described in previous studies [20], the frozen plates with sorted dsDNA-specific single B cells were thawed at room temperature, and reverse transcription was carried out by adding 3 μL of 50 µM random hexamers (Invitrogen), 1 μL of 10 mM dNTP mix (Invitrogen), and 1 μL of SuperScript III (Invitrogen) into each well. The mixture was incubated at 25℃ for 10 min, 50℃ for 50 min, and 85℃ for 5 min. IgH, Igκ, and Igλ genes were amplified from cDNA. All PCRs were performed in 96-well PCR plates in a total volume of 25 μL containing 5 μL of 5 x HotStar HiFidelity PCR Buffer, 1 μL of primer (20 μM), and 0.25 μL of HotStar HiFidelity Polymerase (Qiagen). Each round of PCR was initiated at 94℃ for 5 min, followed by 40 cycles of 94℃ for 30 sec, 58℃ 60 sec, and 72℃ for 1 min, followed by 72℃ for 10 min. The positive products were selected for direct sequencing or subsequently cloned into the corresponding expression vectors as previously described [21]. The recombinant plasmids were co-transfected into 293T cells with equal amounts of paired heavy and light chains using Lipofectamine 3000 (Invitrogen). The full-length of IgGs were expressed and purified using Protein A/G Agarose (ThermoFisher, Waltham, MA).

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