First strand superscript 3 buffer

Manufactured by Thermo Fisher Scientific

The First Strand SuperScript III buffer is a specialized buffer solution designed for use in the first-strand cDNA synthesis step of reverse transcription reactions. It provides the necessary ionic and pH conditions to facilitate the efficient and accurate reverse transcription of RNA templates into complementary DNA (cDNA) strands.

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7 protocols using first strand superscript 3 buffer

Sorting SARS-CoV-2 Spike-Specific B Cells

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Fluorescence-activated cell sorting of cryopreserved PBMCs was performed on a BD FACS Melody. Sorting baits (SARS-CoV-2 Spike) was pre-complexes with the streptavidin flurophore at a 1:4 molar ratio prior to addition to cells. PBMCs were stained with live/dead (fixable Aqua Dead, Thermofisher), anti-CD3-APC/Cy7 (Biolegend), anti-CD8-APC-Cy7 (Biolegend), anti-CD14-BV510 (Biolegend), anti-CD19-PerCP-Cy5.5 (Biolegend), anti-IgM-PE (Biolegend), anti-IgD-Pacific Blue (Biolegend) and anti-IgG-PeCy7 (BD) and Spike-Alexa488 (Thermofisher Scientific, S32354) and Spike-APC (Thermofisher Scientific, S32362). Live CD3/CD8⁻CD14⁻CD19⁺IgM⁻IgD⁻IgG⁺Spike⁺Spike⁺ cells were sorted into individual wells containing RNase OUT (Invitrogen), First Strand SuperScript III buffer, DTT and H₂O (Invitrogen) and RNA was converted into cDNA (SuperScript III Reverse Transcriptase, Invitrogen) using random hexamers following the manufacturer’s protocol.

Graham C., Seow J., Huettner I., Khan H., Kouphou N., Acors S., Winstone H., Pickering S., Galao R.P., Lista M.J., Jimenez-Guardeno J.M., Laing A.G., Wu Y., Joseph M., Muir L., Ng W.M., Duyvesteyn H.M., Zhao Y., Bowden T.A., Shankar-Hari M., Rosa A., Cherepanov P., McCoy L.E., Hayday A.C., Neil S.J., Malim M.H, & Doores K.J. (2021). Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike. bioRxiv.

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Hantavirus Gn-specific B Cell Isolation

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Fluorescence-activated cell sorting of cryopreserved PBMCs was performed on a BD Aria II. PBMCs were stained with anti-rabbit-CD3–phycoerythrin (PE) (Santa Cruz Biotechnology), anti-rabbit-IgM–fluorescein isothiocyanate (FITC) (Southern Biotech), anti-rabbit-IgG–Alexa Fluor 647 (Southern Biotech), and biotinylated HTNV-Gn incubated with streptavidin-peridinin chlorophyll protein (PerCP)-Cy5.5 (BD). CD3⁻ IgM⁻ IgG⁺ HTNV Gn⁺ cells were sorted into individual wells containing RNase OUT (Invitrogen), first-strand SuperScript III buffer, dithiothreitol (DTT), and H₂O (Invitrogen), and RNA was converted into cDNA (SuperScript III reverse transcriptase; Invitrogen) using random hexamers in accordance with the manufacturer’s protocol.

Rissanen I., Krumm S.A., Stass R., Whitaker A., Voss J.E., Bruce E.A., Rothenberger S., Kunz S., Burton D.R., Huiskonen J.T., Botten J.W., Bowden T.A, & Doores K.J. (2021). Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen. mBio, 12(4), e02531-20.

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Characterizing HA-Specific B Cell Responses

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HA-trimer specific B cell response from six healthy human donors was analyzed by flow cytometry. Biotinylated recombinant proteins were conjugated with streptavidin fluorophores resulting in fluorescent labelled-probes. HA-trimers, both WT and RBS-mut, trimeric-stem and monomeric-head were conjugated in an 8:1 ratio (w/w) to streptavidin-conjugates BV421 (0.1 mg/mL, BioLegend, Amsterdam, The Netherlands), BB515 (0.1 mg/mL, BD, Vianen, The Netherlands), or AF647 (0.5 mg/mL, BioLegend) at 4 °C, for 1–2 h.
PBMCs were thawed and stained with the following surface markers: CD3-v500 (UCHT1, BD), CD20-PE-CF594 (2H7, BD), IgM-BV605 (MHM-88, BioLegend), IgG-PE-Cy7 (G18-145, BD), IgA-PE (polyclonal, Southern Biotech), the live/dead marker (viability-eF780, Invitrogen) and streptavidin-conjugated probes at 4 °C, for 30–60 min. Flow cytometry was performed on an ARIA 4 lasers (BD) flow cytometer and the data analysis was performed with FlowJo v10.6.
Live, CD3-, CD20+ and HA-trimer+ cells were single cell sorted into a 96-well plate that contained 20 µL lysis buffer consisting of 20 U RNAse inhibitor (Invitrogen), first strand superscript III buffer (Invitrogen) and 1.25 µL of 0.1 M DTT (Invitrogen). The plates with the sorted cells were stored at −80 °C for at least 24 h before performing the RT-PCR to obtain cDNA.

Aartse A., Eggink D., Claireaux M., van Leeuwen S., Mooij P., Bogers W.M., Sanders R.W., Koopman G, & van Gils M.J. (2021). Influenza A Virus Hemagglutinin Trimer, Head and Stem Proteins Identify and Quantify Different Hemagglutinin-Specific B Cell Subsets in Humans. Vaccines, 9(7), 717.

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SARS-CoV-2 Antigen-Specific B Cell Sorting

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Fluorescence-activated cell sorting of cryopreserved PBMCs was performed on a BD FACS Melody as previously described (Graham et al., 2021 (link)). Sorting baits (SARS-CoV-2 Spike and RBD) was pre-complexed with the streptavidin fluorophore at a 1:4 molar ratio prior to addition to cells. PBMCs were stained with live/dead (fixable Aqua Dead, Thermofisher), anti-CD3-APC/Cy7 (Biolegend), anti-CD8-APC-Cy7 (Biolegend), anti-CD14-BV510 (Biolegend), anti-CD19-PerCP-Cy5.5 (Biolegend), anti-IgM-PE (Biolegend), anti-IgD-Pacific Blue (Biolegend) and anti-IgG-PeCy7 (BD) and Spike-Alexa488 (Thermofisher Scientific, S32354) and Spike-APC (Thermofisher Scientific, S32362) or RBD-Alexa488 and RBD-APC. Live CD3/CD8^-CD14^-CD19⁺IgM^-IgD^-IgG⁺Spike⁺Spike⁺ or CD3/CD8^-CD14^-CD19⁺IgM^-IgD^-IgG⁺RBD⁺RBD⁺ cells were sorted using a BD FACS Melody into individual wells containing RNase OUT (Invitrogen), First Strand SuperScript III buffer, DTT and H₂O (Invitrogen) and RNA was converted into cDNA (SuperScript III Reverse Transcriptase, Invitrogen) using random hexamers (Bioline Reagents Ltd) following the manufacturer’s protocol.

Seow J., Graham C., Hallett S.R., Lechmere T., Maguire T.J., Huettner I., Cox D., Khan H., Pickering S., Roberts R., Waters A., Ward C.C., Mant C., Pitcher M.J., Spencer J., Fox J., Malim M.H, & Doores K.J. (2022). ChAdOx1 nCoV-19 vaccine elicits monoclonal antibodies with cross-neutralizing activity against SARS-CoV-2 viral variants. Cell Reports, 39(5), 110757.

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Splenocyte Isolation and Viral Glycoprotein Analysis

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Fluorescence-activated cell sorting of mouse splenocytes was performed on a BD Aria II. Splenocytes were stained with anti-CD11c-phycoerythrin (PE) (BD Pharmingen), anti-F4/80-PE (eBioscience), anti-CD4-BV605 (BioLegend), anti-CD8-BV605 (BioLegend), anti-B220-BV421 (BioLegend), anti-IgD-allophycocyanin (APC)-Cy7 (BioLegend), and anti-IgM-peridinin chlorophyll protein (PerCP)-eFluor 710 (eBioscience). Biotinylated MACV and JUNV GP1 were incubated separately with both streptavidin-Alexa Fluor 488 (Thermo Fisher Scientific) and streptavidin-Alexa Fluor 647 (Thermo Fisher Scientific). CD4/CD8⁻ B220⁺ IgM⁻ IgD⁻ GP1⁺ cells were sorted into individual wells containing RNase Out (Invitrogen) and First Strand SuperScript III buffer, dithiothreitol (DTT), and H₂O (Invitrogen), and RNA was converted into cDNA (SuperScript III reverse transcriptase; Invitrogen) using random hexamers following the manufacturer’s protocol.

Ng W.M., Sahin M., Krumm S.A., Seow J., Zeltina A., Harlos K., Paesen G.C., Pinschewer D.D., Doores K.J, & Bowden T.A. (2022). Contrasting Modes of New World Arenavirus Neutralization by Immunization-Elicited Monoclonal Antibodies. mBio, 13(2), e02650-21.

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Purification and Characterization of RVFV-Specific B Cells

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PBMCs were purified from rabbit 8315 seven days after the third boost using a Lymphoprep (STEMCELL Technology) density gradient. PBMCs were cryopreserved in FBS plus 10% DMSO. Fluorescence-activated cell sorting of cryopreserved PBMCs was performed. PBMCs were stained with anti-CD3-FITC (Santa Cruz Biotechnology), anti-IgM-PE (Southern Biotech), anti-IgG-PerCP-Cy5.5 (Santa Cruz Biotechnology) and hexahistidine-tagged RVFV-Gn. Cells were washed and anti-HIS-APC (Abcam) was added. CD3^-IgM^-IgG⁺RVFV Gn⁺ cells were sorted into individual wells containing RNase OUT (Invitrogen), First Strand SuperScript III buffer, DTT and H₂O (Invitrogen) and RNA was converted into cDNA (SuperScript III Reverse Transcriptase, Invitrogen) using random hexamers following the manufacturer’s protocol.

Allen E.R., Krumm S.A., Raghwani J., Halldorsson S., Elliott A., Graham V.A., Koudriakova E., Harlos K., Wright D., Warimwe G.M., Brennan B., Huiskonen J.T., Dowall S.D., Elliott R.M., Pybus O.G., Burton D.R., Hewson R., Doores K.J, & Bowden T.A. (2018). A Protective Monoclonal Antibody Targets a Site of Vulnerability on the Surface of Rift Valley Fever Virus. Cell Reports, 25(13), 3750-3758.e4.

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Single-cell RNA-seq of SARS-CoV-2-specific B cells

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SARS-CoV-2 S biotinylated proteins were individually multimerized with fluorescently labeled AF647 and BV421 streptavidin and used for staining as previously described^{13 (link)}. Enriched B cells were stained with probes and antibodies conjugated to fluorophore and LIVE/DEAD dye (ThermoFisher) (Table S1). The lymphocyte population was first gated based on the morphology (FSC-A/SSC-A) and doublets were removed. Dead cells and non-B cells were first excluded within a dump channel (CD3⁻/CD14⁻/CD16⁻). Live B cells (CD19⁺) that were double positive for the SARS-CoV-2 S protein (AF647 and BV421) were single cell-sorted using index sorting into a 96-well plate containing a lysis buffer. The lysis buffer consisted of 20 U RNAse inhibitor (Invitrogen), first strand SuperScript III buffer (Invitrogen), 1.25 μl of 0.1 M DTT (Invitrogen), in a total volume of 20 μL. The plates with the sorted single cells were stored at −80 °C for at least 1 h before performing reverse transcriptase (RT)-PCR.

Claireaux M., Caniels T.G., de Gast M., Han J., Guerra D., Kerster G., van Schaik B.D., Jongejan A., Schriek A.I., Grobben M., Brouwer P.J., van der Straten K., Aldon Y., Capella-Pujol J., Snitselaar J.L., Olijhoek W., Aartse A., Brinkkemper M., Bontjer I., Burger J.A., Poniman M., Bijl T.P., Torres J.L., Copps J., Martin I.C., de Taeye S.W., de Bree G.J., Ward A.B., Sliepen K., van Kampen A.H., Moerland P.D., Sanders R.W, & van Gils M.J. (2022). A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike. Nature Communications, 13, 4539.

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