PBMCs were isolated by density gradient centrifugation using Histopaque (Sigma, Dorset, UK), frozen and stored in liquid nitrogen before staining. Intracellular cytokine staining of Th17 and Th1-cells was carried out using BD Cytofix/Cytoperm kit (BD Bioscience, Oxford, UK). Cells were stimulated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma, Dorset, UK) and 1 μg/mL ionomycin (Sigma, Dorset, UK) for 4 h in the presence of Golgi STOP and Golgi plug. After surface staining using CD3-BV605, CD4-APC and CD8-BV510 antibodies (Biolegend, London, UK), cells were fixed and permeabilised, then stained with IL-17A-FITC (eBiosciences, Ireland, UK) and interferon (IFN)-γ-AF700 (Biolegend). Dead cells were excluded using Fixable Viability Dye eFluor 780 (eBiosciences). Representative FACS plots of the gating strategy and intracellular staining are shown in online supplementary figure S1.
Cd3 bv605
Manufactured by BioLegend
Sourced in United States, United Kingdom
CD3-BV605 is a fluorochrome-conjugated antibody that binds to the CD3 protein complex on the surface of T cells. It is a tool used in flow cytometry analysis for the identification and enumeration of T cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd11c pe cy7, by BioLegend (3 mentions)
Cd4 apc efluor 780, by Thermo Fisher Scientific (3 mentions)
Cd8 bv650, by Thermo Fisher Scientific (3 mentions)
Cxcr3 fitc, by BioLegend (3 mentions)
Lsrfortessa, by BD (3 mentions)
Cd127 bv421, by BioLegend (3 mentions)
Foxp3 staining buffer kit, by Thermo Fisher Scientific (2 mentions)
Cd11b efluor450 m1 70, by Thermo Fisher Scientific (2 mentions)
Cd19 bv711 1d3, by Thermo Fisher Scientific (2 mentions)
Cd4 allophycocyanin efluor780 rm4 5, by Thermo Fisher Scientific (2 mentions)
Klrg1 pe efluor610 2f1, by BioLegend (2 mentions)
Cd127 percp ef710 sb 199, by BioLegend (2 mentions)
Cd11b allophycocyanin cy7, by BioLegend (2 mentions)
Ly6c bv606, by BioLegend (2 mentions)
Cd16 32 fc block, by BD (2 mentions)
Live dead fixable aqua stain, by Thermo Fisher Scientific (2 mentions)
Gata 3 pe, by Thermo Fisher Scientific (2 mentions)
Foxp3 pe cy7, by Thermo Fisher Scientific (2 mentions)
Cd3 fitc 17a2, by Thermo Fisher Scientific (2 mentions)
Siglecf e50 2440, by Thermo Fisher Scientific (2 mentions)
26 protocols using cd3 bv605
1
Intracellular Cytokine Staining of Th17 and Th1 Cells
2
Investigating Immune Checkpoint Modulation in PBMCs
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AIM assays were performed as previously described 43 (link). Briefly, cryopreserved PBMC were thawed, washed, resuspended in R10, and rested for 3 hours at 37°C. Following the 3-hour rest interval, the appropriate number of cells were transferred to a 48-well plate and subsequently treated with CD40 blocking antibody (Miltenyi Biotec, cat. no. 130–094-133) for a final concentration of 0.5ug/mL for 15 minutes at 37°C. Cells were incubated in the presence or absence of our 10-LRA panel, as previously described. After an 18hr incubation, cells were harvested and stained for 50 minutes at 4°C with the surface staining monoclonal antibodies (mAb); (PD-L1-PE/Cy7 (Biolegend, cat. no. 329717), CD40L-PE (BD Biosciences [BD], cat. no. 561720), OX40-APC (BD cat. no. 563473), CD69-BV650 (Biolegend cat. no. 310933), CD3-BV605 (Biolegend cat. no. 317322), CD4-BV421 (BD cat. no. 562424), CD8-PerCp-Cy5.5 (BD cat. no. 560662), CD25- BUV395 (BD cat. no. 564034) and LIVE/DEAD Near-IR stain (ThermoFisher cat. no. L34975), washed, fixed and permeabilized (BD cytofix fixation buffer, cat. no. 554655), and then stained for 30 minutes at 4°C with intracellular mAb Acetyl-histone H3-Alexa Fluor 488 (Cell Signaling Technology, cat. no. 9683S) in 1x perm/wash buffer (BD, cat. no. 554723). The cells were washed and fixed (BD cytofix fixation buffer, cat. no. 554655) prior to flow cytometry analysis.
3
Flow Cytometry Immunophenotyping
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Cells were stained with BD Biosciences (Oxford, U.K.) mAbs: CD45-PerCP-Cy5.5 (30-F11), CD45.2-V450 (104), CD4-BV650 (RM4-5), CD3-FITC (17A2), CD11b-eFluor450 (M1/70), CD19-BV711 (1D3), SiglecF (E50-2440), CD103-PE-CF594 (M290), Ly6G-BV650 (1A8); eBioscience (Loughborough, U.K.) mAbs: CD4-allophycocyanin-eFluor780 (RM4-5); Invitrogen (Dublin, Ireland) mAbs: KLRG1-PE-eFluor610 (2F1) and CD127-PerCP-ef710 (SB/199); and BioLegend (London, U.K.) mAbs: CD45-BV711 (clone: 30-F11), CD3-BV605 (17A2), CD11b-allophycocyanin-Cy7 (M1/70), CD11c-PE-Cy7 (N418), Ly6G-BV785 (1A8), Ly6C-BV606 (HK1.4), and SiglecF-allophycocyanin (S1700L). Before surface staining, Fc receptors were blocked using Fc-Block CD16/32 (BD Biosciences), and cells were incubated with LIVE/DEAD Fixable Aqua stain (Molecular Probes, Invitrogen) to isolate dead cells. For staining of transcription factors, cells were fixed and permeabilized using the Foxp3 staining buffer kit (Invitrogen) and stained with mAbs: GATA3-PE (TWAJ) and Foxp3-PE-Cy7 (FJK-16s). For the detection of YFP, along with intracellular transcription factors from Rora-YFP mice, after surface markers and viability stain, cells were prefixed with 2% paraformaldehyde followed by Foxp3 staining buffer kit. Cells were analyzed using a BD Fortessa (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR), using appropriate controls.
4
VZV-specific CD4+ T cell detection
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HLA-DRB1*15:01 monomers with thrombin-cleavable tethered CLIP peptide and Jun/Fos dimerization domains were produced in insect cells and purified by FPLC. The protein was biotinylated, peptide exchanged with VZV gE peptide 54 (TSPLLRYAAWTGGLA) and VZV IE63 peptide 24 (QRAIERYAGAETAEY) and tetramerized using PE- and APC-labeled streptavidin, respectively. Residual unbound streptavidin and biotinylated DR15 protein was removed from the tetramer product with biotin agarose (Sigma) and streptavidin agarose (ThermoFisher).
Prior to tetramer staining, PBMCs were pre-treated with 50 nM dasatinib (CST) for 30 minutes at 37°C. Tetramer staining (0.5μg/ml for each peptide-exchanged monomer) was performed for one hour at room temperature with Fc block (BioLegend) in 100 μl final volume. During the last 30 minutes of tetramer staining, cells were stained with antibodies to CD38 FITC; CD19, CD8, and γδTCR PerCP-Cy5.5; CCR7 PE-Cy7; CD45RA APC-Cy7; HLA-DR Pacific Blue; CD3 BV605; CD4 BV650 (all BioLegend); and with live/dead aqua zombie. Samples were washed and data were collected using an LSRII flow cytometer (Becton Dickinson). Data analysis was carried out with FlowJo software (TreeStar) gating on single tetramer-positive cells.
Prior to tetramer staining, PBMCs were pre-treated with 50 nM dasatinib (CST) for 30 minutes at 37°C. Tetramer staining (0.5μg/ml for each peptide-exchanged monomer) was performed for one hour at room temperature with Fc block (BioLegend) in 100 μl final volume. During the last 30 minutes of tetramer staining, cells were stained with antibodies to CD38 FITC; CD19, CD8, and γδTCR PerCP-Cy5.5; CCR7 PE-Cy7; CD45RA APC-Cy7; HLA-DR Pacific Blue; CD3 BV605; CD4 BV650 (all BioLegend); and with live/dead aqua zombie. Samples were washed and data were collected using an LSRII flow cytometer (Becton Dickinson). Data analysis was carried out with FlowJo software (TreeStar) gating on single tetramer-positive cells.
5
NK Cell Cytotoxicity Assay with scDbs
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A3.01 and ACH-2 cells (transformed CD4+ T cells, National Institutes of Health (NIH) AIDS Reagents Program, catalog nos. ARP-166 and ARP-349, respectively) were plated in BCM with 10 ng ml−1 of TNF and incubated at 37 °C for 18 h. The cells were washed in BCM to remove TNF. NK cells were isolated from uninfected participant PBMC and cocultured with either the reactivated A3.01 or ACH-2 cells at a 1:3 E:T ratio in BCM at 37 °C for 6 h in the presence of the scDbs or the parental IgG molecules (at the indicated concentrations), monensin (BD Bioscience) and CD107a-BV421 (BioLegend, catalog no. 328625). The supernatants were collected and analyzed by Human CD8/NK Panel Legendplex (BioLegend) per the manufacturer’s protocol. The cells were washed in PBS and stained with the following antibodies (1:50 dilution)/dye (1:1,000 dilution) at 4 °C for 45 min: CD3-BV605 and CD56-FITC (BioLegend; catalog nos. 317321 and 304603, respectively) and Fixable Viability Dye-eFluor780. The cells were washed, and samples were acquired on an Intellicyt iQue Screener Plus. Data were analyzed with FlowJo (see Supplementary Fig. 6 for gating example). NK cells are defined as live CD3−CD56+ lymphocytes. All conditions were tested in triplicate, mean and s.d. shown.
6
Comprehensive Immunophenotyping of Adipose Tissue
7
Immunophenotyping and Isolation of Human T-cells
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Stimulation of cell culture was carried out with 1 μg/ml protein A (SpA, isolated from S. aureus SAC, GE Healthcare, Uppsala, Sweden), 1 µg/ml recombinant SpA (Sigma-Aldrich, Munich, Germany), 100 ng/ml synthetic lipopeptide P3C (Pam3CSK4, EMC, Tübingen, Germany) or anti-CD3/CD28 microbeads (Miltenyi Biotech, Bergisch-Gladbach, Germany).
Microbeads used for cell isolation via AutoMACS and recombinant cytokines (IL-4 and GM-CSF) were obtained from Miltenyi Biotech (Bergisch-Gladbach, Germany).
Antibodies used for flow cytometry, purity determination and cell sorting were purchased from BD Biosciences, Heidelberg, Germany, if not indicated otherwise: CD4 PErCP, CD4 PE, CD3 BV605 (BioLegend, U.S.), CD3 AF700 (BioLegend, U.S.), CD25 APC (BioLegend, U.S.), CD25 FITC, CD127 BV421, CD127 AF647, FoxP3 BV421 (BioLegend, U.S.), CCR4 PE-Cy7 (BioLegend, U.S.), ICOS BV605 (BioLegend, U.S.), CTLA4 BV421 (BioLegend, U.S.), PD-1 PE (BioLegend, U.S.) and CD14 V450. Viability staining was performed with the LIVE/DEAD Fixable dead Cell stain Kit (Thermo Fisher Scientific, U.K.).
Microbeads used for cell isolation via AutoMACS and recombinant cytokines (IL-4 and GM-CSF) were obtained from Miltenyi Biotech (Bergisch-Gladbach, Germany).
Antibodies used for flow cytometry, purity determination and cell sorting were purchased from BD Biosciences, Heidelberg, Germany, if not indicated otherwise: CD4 PErCP, CD4 PE, CD3 BV605 (BioLegend, U.S.), CD3 AF700 (BioLegend, U.S.), CD25 APC (BioLegend, U.S.), CD25 FITC, CD127 BV421, CD127 AF647, FoxP3 BV421 (BioLegend, U.S.), CCR4 PE-Cy7 (BioLegend, U.S.), ICOS BV605 (BioLegend, U.S.), CTLA4 BV421 (BioLegend, U.S.), PD-1 PE (BioLegend, U.S.) and CD14 V450. Viability staining was performed with the LIVE/DEAD Fixable dead Cell stain Kit (Thermo Fisher Scientific, U.K.).
8
Multiparametric Flow Cytometry Immunophenotyping
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Peripheral blood mononuclear cells were prepared for analysis by density centrifugation using Histopaque-1077 (Sigma-Aldrich). The following antibodies were used for flow cytometry immunophenotyping: CD3 – BV605 (Biolegend, San Diego, CA, USA), CD4 – APC-eFluor780 (eBioscience, San Diego, CA,USA), CD8 – BV650 (eBioscience, San Diego, CA,USA), CD25 – PE (eBioscience, San Diego, CA,USA), CD127 – APC (eBioscience, San Diego, CA,USA), CD45RA – PerCP-Cy5.5(eBioscience, San Diego, CA,USA, CD19 – BV450 (BD Bioscience, Franklin Lakes, NJ, USA), CD27 – PE-Cy7 (eBioscience, San Diego, CA,USA, CD62L – APC-eF780 (eBioscience, San Diego, CA,USA, CXCR3 – FITC (Biolegend, San Diego, CA, USA), CXCR5 – AF488 (Biolegend, San Diego, CA, USA), CCR7 – PE (Biolegend, San Diego, CA, USA), PD-1 – APC (eBioscience, San Diego, CA,USA), HLA-DR- eFluor450 (eBioscience, San Diego, CA, USA), IgD – FITC (BD Bioscience, Franklin Lakes, NJ, USA). Flow cytometry analysis was performed on a BD LSRFortessa (BD Bioscience) with FACS Diva software (BD Bioscience) for acquisition, then analysis was performed with FlowJo software (LLC).
9
Comprehensive Immune Response Profiling
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IVS-expanded PBMCs were stimulated with either minimal (8-11mer) or long overlapping (15mer) peptides, 20% (v/v) H2O and 80% (v/v) DMSO (vehicle control; VWR) or PMA/Ionomycin cell stimulation cocktail (positive control; Affymetrix, Santa Clara, CA, USA) in the presence of anti-Human CD28/CD49d antibody (BD Biosciences, San Jose, CA, USA), BD GolgiStop (BD Biosciences), and Brefeldin A (BioLegend) over a period of 18 h. Following overnight incubation, cells were stained with live/dead Zombie-Red (BioLegend, 1:500 dilution) and surface marker antibodies (CD8-PerCP-Cy5.5 (1:200), CD3-BV605 (1:100), from BioLegend; CD4-APC-eF780 (1:100) from eBioscience) prior to fixation and permeabilization with FIX & PERM™ Cell Permeabilization Kit (ThermoFisher, Waltham, MA, USA). Following permeabilization, cells were stained for intracellular cytokines using anti-human IFNγ-APC (1:50), TNFα-BV785 (1:50), and IL-2-PE (1:25) antibodies (BioLegend) prior to data acquisition on a BD LSRFortessaTM flow cytometer (BD Biosciences) using FACSDivaTM Software version 9.2.
10
NK Cell Activation by Dendritic Cells
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NK cells were enriched from PBMCs by the depletion of unwanted cells using biotinylated antibodies and MoJo Streptavidin Nanobeads (BioLegend); more details are available in SI Appendix . The enriched NK cells were stained with CD3-BV605 (clone: UCHT-1, BioLegend), CD19-BV605 (clone: SJ25C1, BioLegend), CD20-BV605 (clone: 2H7, BioLegend), HLA-DR-BV510 (clone: L243, BioLegend), CD56-BV421 (clone: 5.1H11, BioLegend) and NKp46-BV421 (clone: 9E2, BioLegend) and sorted as CD56+NKp46+CD3−CD19−CD20−HLA-DR− cells. Sorted XCR1− and XCR1+ cDC1 and NK cells were cocultured in a 1:5 ratio and either stimulated with a cocktail of pIC (2.5 µg/mL), R848 (2.5 µg/mL), and CpG (2.5 µg/mL) or cultured in DC-medium alone. For some experiments, DCs and NK cells were separated in Transwell plates (96 well, 0.4 µm insert). NK cells were cultured in the well, while DCs were added to insert. In order to analyze the influence of secreted cytokines on NK cells, NK cells were cultured alone and supernatants of stimulated DCs were added corresponding to a 1:5 DC:NK cell ratio. After 18 h, cells were harvested and analyzed by flow cytometry for activation of NK cells (CD69). The supernatants were stored at −80 °C until analysis with LEGENDplex Human Interferon Panel (BioLegend) for secretion of IFNγ.
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