Frozen splenocytes were thawed and incubated with 50 μg/mL DNase1 (Cat# 10104159001, Roche) for 5 min at 37°C. The cells were then washed twice and resuspended in RPMI-1640 medium with 10% (vol./vol.) heat-inactivated FBS. Splenocytes (1×106/well in 96-well plates) were stimulated for 24 h with 15-mer overlapping peptides from SARS-CoV-2 spike glycoprotein (Cat no# PM-WCPV-S-1, JPT Peptide Technologies GmbH) or VSV-N (Genscript) at a concentration of 2.5 μg/mL. Supernatants were collected, centrifuged at 1,800 RPM for 5 min and stored at −80 °C until analysis. Supernatants were then analyzed for the expression of IFN-γ, IL-6, IL-18, GM-CSF, IL-1β, IL12p70, IL-13, IL-2, IL-4, TNF-α and IL-5 cytokines using a mouse cytokine 11-plex antibody bead kit (Th1/Th2 Cytokine 11-Plex Mouse ProcartaPlex™ Panel, Cat No# EPX110-20820-901, Thermo Fisher). Preparation of samples, along with kit standards, detection antibody and streptavidin-phycoerythrin (PE), was carried out per the manufacturer’s instructions. Cytokine bead fluorescence intensity was measured using the Luminex 200 system (Luminex Corp., Austin, TX, USA), and data were quantitated with xPONENT® software.
Pm wcpv s 1
Manufactured by JPT Peptide Technologies
Sourced in Germany
The PM-WCPV-S-1 is a freeze dryer or lyophilizer designed for laboratory use. It is capable of freeze-drying small sample volumes. The device features a stainless-steel chamber and shelves, and can maintain precise temperature and pressure conditions during the freeze-drying process.
Automatically generated - may contain errors
Lab products found in correlation
Vsv n, by GenScript (2 mentions)
Dnase 1, by Roche (2 mentions)
Anti cd4 fitc, by BioLegend (1 mentions)
Golgistop monensin, by BD (1 mentions)
Anti cd3 apc cy7, by BioLegend (1 mentions)
Pm wcpv ncap 1, by JPT Peptide Technologies (1 mentions)
Anti cd8 percp, by BioLegend (1 mentions)
Anti ifn γ pe 4 s b3, by BioLegend (1 mentions)
Facslyric, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
4 protocols using pm wcpv s 1
1
Cytokine Profiling of SARS-CoV-2 Spike Peptide-Stimulated Splenocytes
2
SARS-CoV-2 Antigen-Specific T Cell Assay
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Virus-specific CD8+ T-cell frequencies were measured by flow cytometric analysis of gamma interferon (IFN-γ) induction after specific stimulation as described previously [42 (link)]. PBMCs were prepared from whole blood by density gradient centrifugation using Ficoll-Paque PLUS (Cytiva). Lymph node-derived lymphocytes were prepared from minced lymph nodes by density gradient centrifugation using Ficoll-Paque PLUS. Cells were pulsed and cocultured with peptide pools (at a final concentration of more than 0.1 μM for each peptide) using panels of overlapping peptides spanning the SARS-CoV-2 S, N, M, and E amino acid sequences (PM-WCPV-S-1, PM-WCPV-NCAP-1, PM-WCPV-VME-1, and PM-WCPV-VEMP-1; JPT Peptide Technologies) in the presence of GolgiStop (monensin, BD), 1 μg/ml of anti-CD28 (CD28.2, BD) and 1 μg/ml anti-CD49d (9F10, BD) for 6 hours. Intracellular IFN-γ staining was performed with a CytofixCytoperm kit (BD) and anti-CD3 APC-Cy7, anti-CD4 FITC, anti-CD8 PerCP, and anti-IFN-γ PE (4S.B3; BioLegend). Stained cells were analyzed by BD FACS Lyric. A representative gating schema for flow cytometric analysis is shown in Fig 5A . Specific T-cell frequencies were calculated by subtracting nonspecific IFN-γ+ T-cell frequencies from those after peptide-specific stimulation. Specific T-cell frequencies less than 0.03% of CD8+ T cells were considered negative.
3
Enrichment of Antigen-Specific T Cells
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Peripheral blood mononuclear cells (PBMCs) from leukaphereses and buffy coats were isolated as previously described (20 (link)). The isolated PBMCs were then cryopreserved in liquid nitrogen. The cryopreserved cells were reconstituted, and a 14-day enrichment with antigen-specific T cells was performed as previously described (21 (link)). For the enrichment of the reconstituted cells with antigen-specific T cells, a 1 μg/ml concentration of the following pooled overlapping peptide mixes spanning the indicated antigen was used: SARS-CoV-2 (17 (link)) [PepMix™ SARS-CoV-2 (Spike Glycoprotein), cat.# PM-WCPV-S-1, JPT Peptide Technologies, Berlin, Germany], and human coronavirus 229E (18 (link)) [PepMix™ HCoV-229E (Spike Glycoprotein), cat.# PM-229E-S-1, JPT]. As a positive control, pooled peptide mixes from Epstein-Barr virus (HHV-4), human cytomegalovirus (HHV-5), and influenza A (22 (link)) were used [1 μg/ml, PepMix CEF Pool (extended), cat.# PM-CEF-E, JPT].
4
Profiling SARS-CoV-2 Spike Protein Immunity
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Frozen splenocytes were thawed and incubated with 50 µg/mL DNase1 (Cat# 10104159001, Roche) for 5 min at 37°C. The cells were then washed twice and resuspended in RPMI-1640 medium with 10% (vol/vol) heat-inactivated FBS. Splenocytes (1 × 106/well in 96-well plates) were stimulated for 24 h with 15-mer overlapping peptides from SARS-CoV-2 spike glycoprotein (Cat no# PM-WCPV-S-1, JPT Peptide Technologies GmbH) or VSV-N (Genscript) at a concentration of 2.5 µg/mL. Supernatants were collected, centrifuged at 1,800 RPM for 5 min, and stored at −80°C until analysis. Supernatants were then analyzed for the expression of IFN-γ, IL-6, IL-18, GM-CSF, IL-1β, IL12p70, IL-13, IL-2, IL-4, TNF-α, and IL-5 cytokines using a Mouse Cytokine 11-Plex Antibody Bead Kit (Th1/Th2 Cytokine 11-Plex Mouse ProcartaPlex Panel, Cat No# EPX110-20820-901, Thermo Fisher). Sample preparation, along with kit standards, detection antibody, and streptavidin-phycoerythrin, was carried out according to the manufacturer’s instructions. Cytokine bead fluorescence intensity was measured using the Luminex 200 system (Luminex Corp., Austin, TX, USA), and data were quantitated with xPONENT software.
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