Cryopreserved PBMCs were thawed, resuspended in 10 ml of RPMI 10% FCS, and collected by centrifugation at 600 × g for 5 min. Cells were washed with PBS and resuspended in 10 ml of PBS and collected by a second centrifugation step. Cells were resuspended in 100 μl of FWB (2% FCS PBS) with anti-rabbit IgM FITC (1:1000) and a streptavidin-APC tetramer of biotinylated anti-rabbit IgG. After 1 h on ice, cells were washed once with 10 ml of PBS, collected by centrifugation, and resuspended in 100 μl of FWB with 1 μl of a streptavidin-PE tetramer of biotinylated BG505 SOSIP.664 and 1 μl of a mixed streptavidin-BV786 tetramer of B41 and ZM197 SOSIP.664. After a further 1 h on ice, cells were washed once with 10 ml of PBS, collected by centrifugation and resuspended in 500 μl of FWB for sorting on a BD FACS Aria III. IgM-IgG+BG505+B41+ZM197+ lymphocytes were collected at 1 cell per well into Superscript III Reverse Transcriptase lysis buffer (Invitrogen) as previously described and immediately stored at −80°C prior to cDNA generation and single cell PCR (see Table S1 ).
Superscript 3 reverse transcriptase lysis buffer
Manufactured by Thermo Fisher Scientific
Superscript III Reverse Transcriptase lysis buffer is a buffer solution used in the process of reverse transcription, which is the conversion of RNA into complementary DNA (cDNA). The buffer is designed to facilitate the activity of the Superscript III reverse transcriptase enzyme, which is responsible for the reverse transcription reaction.
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Lab products found in correlation
2 protocols using superscript 3 reverse transcriptase lysis buffer
1
Isolation and Characterization of Antigen-Specific B Cells
2
Single-cell analysis of HIV-neutralizing antibodies
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Cryopreserved PBMCs from rabbit 5743 were thawed, resuspended in 10 ml of RPMI 10% fetal calf serum (FCS), and collected by centrifugation at 600g for 5 min. Cells were washed with phosphate-buffered saline (PBS), resuspended in 10 ml of PBS, and collected by a second centrifugation step. Cells were resuspended in 100 μl of FWB (2% FCS–PBS) with anti-rabbit immunoglobulin M (IgM) fluorescein isothiocyanate (FITC) (1:1000) and a streptavidin-allophycocyanin (APC) tetramer of biotinylated anti-rabbit IgG. After 1 hour on ice, cells were washed once with 10 ml of PBS, collected by centrifugation, and resuspended in 100 μl of FWB with 1 μl of a streptavidin-phycoerythrin (PE) tetramer of biotinylated BG505 or B41 SOSIP.664. After a further 1 hour on ice, cells were washed once with 10 ml of PBS, collected by centrifugation, and resuspended in 500 μl of FWB for sorting on BD FACSAria III. IgM−IgG+BG505+B41 lymphocytes were collected at one cell per well into SuperScript III Reverse Transcriptase lysis buffer (Invitrogen) as previously described and immediately stored at −80°C before complementary DNA generation and single-cell polymerase chain reaction.
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