The generation of the different SARS-Cov-2-specific CD4+ T cell lines and their specificity and reactivity were confirmed by an immunophenotypic and functional assay where the cell lines were stimulated overnight in 5% CO2 at 37°C with their irradiated autologous feeder cells in the absence (media) or in the presence of their respective antigens (1µg/mL of MP-S or 1µg/mL of MP-M). Both CD4+ TCLs were also stimulated with 1µg/mL MP-CMV and PMA/ionomycin (Sigma-Aldrich) (0.5/0.05 pg/ml). The cells were then stained for viability (Live/Dead fixable blue (UV450), Molecular Probes), and then incubated with anti-CD3 (BV421), anti-CD4 (PerCP-Cy5.5) for 30 min in the dark at room temperature. The cells were next washed twice with FACS buffer, then fixed and permeabilized using a Fix/Perm buffer kit (BioLegend) for 30 min at 4°C. The cells were washed twice with Perm buffer (BioLegend) and resuspended with the intracellular antibody pool containing anti-CD69 (FITC), anti-CD154 (APC), anti-TNF-α (Alexa Fluor 700) and anti-IFN-γ (BUV737) for 30 min at 4°C. Finally, the cells were washed twice with Perm buffer and then acquired using the BD LSRFortessa flow cytometer (BD Biosciences) and FACSDiva software (BD Biosciences) for acquisition. All analyses were performed using FlowJo v10.5.3.
Fix perm buffer kit
Manufactured by BioLegend
The Fix/Perm buffer kit is a laboratory solution designed for the fixation and permeabilization of cells for flow cytometry analysis. The kit contains the necessary buffers to prepare samples for intracellular staining and flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (2 mentions)
Pma ionomycin, by Merck Group (2 mentions)
Perm buffer, by BioLegend (2 mentions)
Perm buffer, by BD (2 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Anti tnf α, by BioLegend (1 mentions)
Anti ifn γ, by BioLegend (1 mentions)
Facsaria 2, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Pe anti mouse cd86, by BioLegend (1 mentions)
Apc anti mouse cd206, by BioLegend (1 mentions)
Fitc anti human cd206, by BioLegend (1 mentions)
Apc anti human cd86, by BioLegend (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Lsrfortessa x 50 flow cytometer, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
4 protocols using fix perm buffer kit
1
Characterization of SARS-CoV-2-specific CD4+ T cells
2
Profiling NK2R expression in DLD-1 cells
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DLD‐1 cells were treated with a Fix/Perm Buffer kit (Biolegend) and stained with anti‐NK2R mAb. The expression of NK2R was evaluated by FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star) according to the previous study.27 In some animal model experiments, CD45+CD11c+7AAD− cells and CD45−GFP+7AAD− CT26 cells were isolated as DCs and colon cancer cells by FACSAria II (BD Biosciences), respectively.
3
Multicolor Flow Cytometry for Cell Markers
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For cell marker flow staining, cells were harvested and resuspended in FACS buffer (PBS contained 2% FBS). Then, the cells were blocked with Fc Receptor block solution (Biolegend, London, UK, Human TruStain FcX, 422301; anti-mouse CD16/32 antibody, 156603) and incubated with cell surface antibodies (CD86) on ice for 20 min. Following surface staining, cells were fixed and then permeabilized using a Fix/Perm buffer kit (BioLegend, 426803) and subsequently stained with intracellular antibodies (CD206). Cells were washed, resuspended in FACS buffer, and tested on CytoFlex (Beckman, USA). Flow antibodies were as follows: APC anti-human CD86 (Biolegend, 374207), FITC anti-human CD206 (Biolegend, 321103), APC anti-mouse CD206 (Biolegend, 141707), and PE anti-mouse CD86 (Biolegend, 129203).
4
Comprehensive Immunophenotypic Profiling of PBMCs
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Comprehensive immunophenotypic characterization was performed on 1 × 106 PBMCs of all 47 individuals at baseline (media alone), or after stimulation with PMA/ionomycin (0.5/0.05 pg/mL; Sigma-Aldrich) for 12 hours, in the presence of brefeldin A (10 μg/mL; Sigma-Aldrich), by extra- and intracellular staining using a 27-multiparameter flow cytometry panel. After stimulation, cells were stained for viability (Live/Dead fixable blue, Molecular Probes) for 10 minutes in the dark at room temperature, washed with FACS buffer, and then incubated with a fluorochrome-conjugated antibody’s master mix for surface markers (Supplemental Table 1 ) for 30 minutes in the dark at room temperature. The cells were next washed twice with FACS buffer, and then fixed and permeabilized using a Fix/Perm buffer kit (BioLegend) for 30 minutes in the dark at 4°C. The cells were washed twice with Perm buffer (BioLegend) and resuspended with fluorochrome-conjugated antibody’s master mix for intracellular markers (Supplemental Table 1 ) for 30 minutes in the dark at 4°C. Finally, the cells were washed twice with Perm buffer and then acquired using the LSRFortessa X50 flow cytometer (BD Biosciences) and FACSDiva software (BD Biosciences) for acquisition. All analyses were performed using FlowJo v.10.5.3.
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