Thawed PBMCs were first stained with Decoy tetramer and then with RBD tetramer prior to incubation with anti-PE magnetic beads and magnetic bead enrichment (Miltenyi Biotec) as previously described.17 (link) Bound cells were stained with surface antibodies (SI Table 1 ) and, if required, were fixed/permeabilized using eBioscience FoxP3 Fix/Perm kit (ThermoFisher; 00-5521-00) for 30 minutes, followed by incubation with intracellular antibodies (SI Table 1 ). Stained samples were run on a LSRII flow cytometer and analyzed using FlowJo (Becton Dickinson). For B cells sorting experiments, single tetramer-specific B cells were indexed sorted on a FACSAriaII cell sorter and collected in a 96-well PCR plate containing SMART-Seq v4 capture buffer (Takara Bio).
Smart seq v4 capture buffer
Manufactured by Takara Bio
The SMART-Seq v4 capture buffer is a reagent used in the SMART-Seq v4 RNA-seq protocol. It serves to capture and reverse transcribe RNA molecules into cDNA.
Automatically generated - may contain errors
3 protocols using smart seq v4 capture buffer
1
SARS-CoV-2 RBD-specific B cell isolation
2
Isolation and Sequencing of SARS-CoV-2 Specific B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood samples from convalescent COVID-19 subjects were obtained with informed consent under the approval of the University of Washington Institutional Review Board (Gale Lab; 00009810) and isolated as previously described (Rodda et al., 2021 (link)). Briefly, peripheral blood mononuclear cells were thawed, washed, and stained with a decoy tetramer and then RBD tetramer; bound cells were magnetically enriched (Miltenyi Biotec) and stained with surface antibodies. Single tetramer-positive B cells were index-sorted using a FACS Aria II and collected in 96-well plates containing SMART-Seq v4 capture buffer (Takara Bio) for BCR sequencing.
3
Enrichment and Phenotyping of SARS-CoV-2 RBD-Specific B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thawed PBMCs were first stained with decoy tetramer and then with RBD tetramer prior to incubation with anti-PE magnetic beads and magnetic bead enrichment (Miltenyi Biotec) as previously described.17 Bound cells were stained with surface antibodies and, if required, were fixed/permeabilized using eBioscience FoxP3 Fix/Perm kit (ThermoFisher) for 30 min, followed by incubation with intracellular antibodies. Stained samples were run on a LSRII flow cytometer and analyzed using FlowJo (Becton Dickinson). Samples with less than 5 RBD-specific MBCs were removed from summary data for proportional phenotyping (surface stain V1: 2, surface stain V2: 1, intracellular stain V1: 4, intracellular stain V2: 3). For B cells sorting experiments, single tetramer-specific B cells were indexed sorted on a FACSAriaII cell sorter and collected in a 96-well PCR plate containing SMART-Seq v4 capture buffer (Takara Bio).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!