Immunofluorescence staining was performed using the following antibodies at the stated concentrations: BioLegend: CD3-BV421 (SK7; 1:25), CD4-AF594 (RPA-T4; 1:50), CD19–AF647 (SJ25C1; 1:25), CXCR4 (12G5; 1:100), CD21-FITC (Bu32; 1:100), PD-1-AF488 (NAT105; 1:100), BCL-6 (IG191E/A8; 1:50); Sigma-Aldrich: CXCR5 (Polyclonal; 1:100), CD83 (Polyclonal; 1:100); Thermo Fisher: AID (ZA001; 1:100), CD8-AF488 (AMC908; 1:100), CD20-eFluor 615 (L26; 1:100), CD20-eFluor 660 (L26; 1:100); BD Biosciences: Ki67-BV421 (B56; 1:5); Fisher Science: IgD-AF488 (IgD26; 1:10).
Cd3 bv421
Manufactured by BioLegend
Sourced in United States
CD3-BV421 is a fluorochrome-conjugated antibody that binds to the CD3 antigen, a cell surface marker expressed on T cells. It is designed for use in flow cytometry applications to identify and quantify T cell populations in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cd4 fitc, by BioLegend (2 mentions)
Cd19 bv605, by BioLegend (2 mentions)
Cd8 pe cy7, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Cd8 pe, by BioLegend (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Cxcr4, by Merck Group (1 mentions)
Bcl 6, by Merck Group (1 mentions)
Cd45r pe, by BioLegend (1 mentions)
Cd44 bv605, by BioLegend (1 mentions)
Cd62l pe cf594, by BD (1 mentions)
Cd4 apcef780, by Thermo Fisher Scientific (1 mentions)
Cd69 fitc, by Thermo Fisher Scientific (1 mentions)
Il 17a percp cy5, by Thermo Fisher Scientific (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
20 protocols using cd3 bv421
1
Multiparametric Immunofluorescence Profiling
2
Comprehensive Immune Cell Profiling
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Lung and nasal tissue mononuclear cell suspensions were prepared by mechanical (chopping with a scalpel) followed by enzymatic disruption of tissue for 1 h at 37°C with Collagenase D (1 mg/ml; Sigma-Aldrich) and DNAse I (20 U/ml; Sigma-Aldrich). Next, lungs or spleens were passed through a 40-mm cell strainer to a obtain single-cell suspension, followed by RBC lysis. The cells were incubated with CD16/CD32 FcgRIII (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. To detect cytokines, cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (5 mg/ml) for 4 h at 37°C. The following surface Abs were used: CD45R-PE, CD3-BV421, CD44-BV605 (Biolegend), CD62L-PE-CF594 (BD), CD103-BV786, CD4-APC-eF780, CD69-FITC (eBioscience). For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–PerCP-Cy5.5 and IFN-γ-BV650 (eBiosciences). Fluorescence minus one or non-specific isotype Abs were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar).
3
Comprehensive T-cell Signaling Antibody Panel
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Antibodies and reagents used for flow cytometry were: CD8-APC, Fixable viability stain 450, antiCD16/32 (eBiosciences); anti-rabbit-PE (Jackson Immunoresearch); anti-SLP76 (pY128), CD4-PE, CD25-FITC, CD8 FITC and anti-mouse IgG1-PE (BD Biosciences); CD4-PEVio770 (Miltenyi Biotec); CD3-BV421 (Biolegend); anti-pErk1/2 (pT202/pY204) (Cell signaling Technology). The following reagents were used for cell stimulation, immunoblotting or immunoprecipitation: anti-CD3-biontinylated, anti-CD28-biontinylated, anti-CD3ε, anti-CD28 (eBiosciences); streptavidin (Sigma); anti-pErk1/2 (pT202/pY204), anti-pPLCγ1 (pY783), anti-pp38 (pT180/pY182), anti-pJNK (pT183/Y185), anti-pAkt (pS473), anti-pSLP76 (pS376) (Cell Signaling Technology); anti-GADS (Santa Cruz Biotechnology Inc.); rabbit anti-SLP76 or goat-anti-SLP76 (Thermo Fisher Scientific); anti-β-tubulin (Chemicon); for anti-14-3-3 immunoblotting we used a mix of anti-pan 14-3-3 and anti-14-3-3ζ (Santa Cruz, Biotechnology Inc.).
4
Flow Cytometry Antibody Panel
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The following antibodies were used in flow cytometry: B220 FITC 1:50 (BD Pharmingen, RA3-6B2), CD3 BV421 1:200 (17A2, BioLegend), CD3 FITC 1:50 (17A2, BD Pharmingen), CD3 PE-Cy-7 1:25 (145-2C11, BD Pharmingen), CD11b PerCP-Cy5.5 1:400 (M1/70, eBioscience), CD11b BV421 1:400 (M1/70, BioLegend), CD11b PE-Cy-7 1:400 (M1/70, eBioscience), CD11c BV421 1:100 (N418, BioLegend), CD16/32 unconjugated 10 μg ml−1 (93, BioLegend), CD19 BV421 (6D5, BioLegend), CD19 APC 1:400 (1D3, BD Pharmingen), CD45 BV421 1:400 (30-F11, BioLegend), CD45 BV785 1:400 (30-F11, BioLegend), CD49b APC 1:100 (DX5, BD Pharmingen), CD90.2 APC-Cy7 1:400 (30-H12, BioLegend), CD117 PE 1:800 (2B8, eBioscience), CD117 APC 1:800 (2B8, BD Pharmingen), CD117 BV711 1:800 (2B8, BioLegend), FcεRI APC 1:200 (MAR-1, eBioscience), Gr-1 BV421 1:800 (RB6-8C5, BioLegend), Gr-1 BV605 1:200 (RB6-8C5, BioLegend), IgE PE 1:100 (RME1, BioLegend), IgE BV786 1:100 (RME-1, BD Pharmingen), IgE BV421 1:100 (RME-1, BD Pharmingen), Ly6G PerCP-Cy5.5 1:100 (1A8, BD Pharmingen), MHCII A700 1:100 (M5/114.15.2, eBioscience), Siglec-F BV421 1:100 (E50-2440, BD Pharmingen), Siglec-F PE 1:100 (E50-2440, BD Pharmingen), Ter119 BV421 1:200 (Ter119, BioLegend), 5-HT unconjugated 0.11 μg ml−1 (5HT-H209, Dako) and mouse-IgG1 PE 1:100 (RMG1-1, BioLegend).
5
Purifying MAGE-A3-Specific T Cell Cultures
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Sorting was used to purify αβTCR-specific cultures and was performed on the FACS Aria(BD bioscience). For sorting, the MAGE-A3 transduced T cell cultures were first stained with HLA-A1 tetramers for 30 minutes at 37°C. For that, 20 ng of PE- and APC-conjugated MAGE-A3 specific tetramers were added to1 x 106 T cells in 50 mL of 1x PBS, 0.5% bovine serum albumin (BSA, Sigma Aldrich) and 2 mM EDTA. After the tetramer staining, CD3-BV421 (Biolegend), in a volume of 50 µl, was added directly into the tetramer/cell mix and incubated for 20–30 minutes in the dark and on ice. The transduced MAGE-A3 (also named MAGE-A3a3a) tetramer positive T cells were sorted directly into a rapid expansion protocol (REP) for further culturing. All antibodies, buffers and procedures were kept under sterile conditions to ensure aseptic sorting of cells for further culturing.
6
Comprehensive Tumor Immune Profiling
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4T1 tumors were excised and digested with the Mouse Tumor Dissociation kit (Miltenyi Biotec) as per manufacturer’s instructions, and ran on a Miltenyi gentleMACS Octo Dissociator with Heaters using pre-set program (37C_m_TDK2). The resulting cell suspensions were filtered using a 40 µm cell strainer and subjected to RBC lysis. Samples were counted and stained with the Zombie Aqua Fixable Viability Dye (BioLegend) to distinguish live cells. All samples were then incubated with purified anti-mouse CD16/32 (Fc block) prior to staining. The following anti-mouse antibodies, all purchased from BioLegend, were used for immunostaining in the indicated dilutions: CD69 APC (Clone H1.2 F3) 1:100, CD4 PE/Cy5 (Clone GK1.5) 1:100, FOXP3 Alexa Fluor 488 (Clone 150D) 1:50, CD25 APC (Clone PC61) 1:100, CD45 APC-Cy7 Clone 30-F11 (1:500), CD3 BV421 Clone 145–2 C11(1:100), CD8 FITC Clone 53–6.7 (1:100), CD11b PerCP-Cy5.5 Clone M1/70 (1:200), Ly6G Alexa Fluor 488 (Clone 1A8) 1:100, Ly6C Brilliant Violet 421 (Clone HK1.4) 1:100, CD4 PerCP-Cy5.5 Clone GK1.5 (1:100). Flow data were acquired using a MACSQuant Analyzer 10 and analyzed using FlowJo version 10.1 (Tree Star).
7
Identification of Th17 Cells and ILC3s
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Prior to staining with primary antibodies, cells were incubated in RPMI media containing 10% FBS, 50 ng/mL PMA, 1 μg/mL ionomycin and brefeldin A (1:1000; Biolegend) for 4 h at 37°C. Following staining with primary antibodies in PBS containing 3% FBS, cells were fixed using 4% paraformaldehyde, permeabilized, and stained overnight in permeabilization buffer. The primary antibodies were diluted as described: CD45-A700 (1:100; Biolegend, Clone: 30-F11), CD4-FITC (1:100; Biolegend, Clone: GK15), CD3-BV421 (1:100; Biolegend, Clone: 17A2), Lin-biotin conjugated (1:50; Miltenyi Biotec: cocktail containing antibodies against CD5, CD11b, CD45R, anti-7-4, anti-Gr-1 and anti-Ter119), Streptavidin-PE-CF594 (1:500; BD Biosciences), IL-17A-PE-Cy7 (1:1000; eBioscience, Clone: eBio17B7), and IL-22-APC (1:100; eBioscience, Clone: IL22JOP). Th17 cells were categorized as CD45+CD3+CD4+IL-17A+, while ILC3s were categorized as CD45+Lin−CD3−IL-17A+IL-22+. The gating strategy is described in detail in Supplementary Figure 1 .
8
Immune cell profiling of quadriceps muscle
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Perfused ipsilateral quadriceps muscles were minced and digested in RPMI supplemented with 10% HI-FBS, HEPES pH 7.3, 100 U/ml penicillin and streptomycin, collagenase (2.5 mg/ml; Sigma), and DNase I (30 μg/ml; Roche) in a total of 5 ml at 37°C for 1.5 h. Digested tissue was pressed through a 70 μm cell strainer, and resuspended in RPMI supplemented with 10% HI-FBS and penicillin and streptomycin. Cells were counted using precision count beads (BioLegend). Single cell suspensions were blocked for FcγR binding (BioLegend; clone S17011E) and stained with the following antibodies: CD3 BV421 (BioLegend; clone 145-2C11), CD4 FITC (BioLegend; clone RM4-5), CD8α APC (BioLegend; clone 53–6.3), NK1.1 PE (BioLegend; clone PK136), CD45 BUV395 (BD Biosciences; clone 30-F11), CD19 BV605 (BioLegend; clone 6D5), CD11b PerCP-Cy5.5 (BioLegend; clone M1/70), Ly6C Pacific Blue (BioLegend; clone HK1.4), Ly6G phycoerythrin (PE)-Cy7 (BioLegend; clone 1A8), MHC class II Alexa Fluor 700 (BioLegend; clone M5/114.15.2), Ly6B FITC (BioLegend; clone 7/4), and F4/80 BV650 (BioLegend; clone BM8). Viability was determined by exclusion of a fixable viability dye (eBiosciences; e506). Samples were run on a BD LSRFortessa X-20 flow cytometer and analyzed using FlowJo version 10 (FlowJo, LLC).
9
Flow Cytometry Analysis of PBMCs
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The mAbs used for cell surface staining were CD3-APCA750, CD14-PE, CD16-APC, CD4-APC (all from Beckman Coulter, Villepinte, France) and CD3-BV421 and CD3-AF700 (both from Biolegend, Paris, France). Annexin V–PE (Biolegend) was used according to the manufacturer’s guidelines, and the labeling was analyzed at day 6. For ROS quantification, 106 PBMCs were resuspended in 1 μM dichlorodihydrofluorescein diacetate (DCFH-DA) for 25 minutes at room temperature. Data were acquired on a Navios flow cytometer (Beckman Coulter) from 20,000 gated events per sample and on a MACSQuant analyzer 10 (Miltenyi Biotech) and analyzed using Kaluza software.
10
Comprehensive Immune Cell Profiling by Flow Cytometry
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Flow cytometry data was collected on a FACS Fortessa (BD) and analyzed using FlowJo (Treestar). The following antibodies and reagents were used: LIVE/DEAD Fixable Dead Cell Stain (Invitrogen), CD3-BV421 (BioLegend), CD4-BV711 (BioLegend), CD8-BV605 (BioLegend), CD19-FITC (BD Pharmingen), CD69-PE-Cy7 (BD Pharmingen), CD25-PE-CF594, (BD Horizon), Annexin V-APC (BD Pharmingen), and propidium iodide (PI) (BD Pharmingen). For proliferation and activation assays, cells were washed once with PBS, and then incubated with LIVE/DEAD Fixable Dead Cell Stain in PBS for 30 minutes at room temperature in the dark. After washing with PBS with 1% FBS, cells were incubated with surface marker antibodies in PBS with 1% FBS for 20 minutes at room temperature in the dark. The cells were then washed and resuspended in PBS with 1% FBS, and the samples were run on the flow cytometer. For Annexin V and PI staining, cells were washed once with binding buffer (eBioscience), then incubated with Annexin V and cell surface markers in binding buffer for 15 minutes at room temperature in the dark. After washing with binding buffer, cells were resuspended in binding buffer with PI, and the samples were analyzed by flow cytometry.
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