For flow cytometry or cell sorting, cells were transferred to 15-mL tubes and washed in cold buffer (2% FCS, 2 mM EDTA in PBS). Before antibody labeling, cells were incubated with Fc-Block (1:200, anti-CD16/32 antibody; 2.4G2, BD Pharmingen) for 10 min at 4°C and washed. Cells were stained in buffer with (1:100) fluorescently labeled antibodies against CD11b (clone M1/70, BD Pharmingen), CD11c (clone N418, Biolegend), CD115 (clone AFS98, eBioscience), CD19 (clone 1D3, BD Pharmingen), and Ly-6G (1A8, Biolegend) for 30 min at 4°C in the dark. 7-AAD (BD Pharmingen) was added before measurement to exclude dead cells. Analysis was performed on LSRFortessa and sorting on FACSAria II or III (BD Pharmingen). Unstained, empty vector-transduced cells or fluorescence-minus-one staining setups served as controls.
Cd19 clone 1d3
Manufactured by BD
Sourced in United States, Germany
CD19 (clone 1D3) is a lab equipment product designed for the detection and analysis of CD19 cell surface antigen. It is a monoclonal antibody that specifically binds to the CD19 protein expressed on the surface of B cells and B cell precursors.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b clone m1 70, by BD (3 mentions)
Ly6c clone al 21, by BD (3 mentions)
B220 clone ra3 6b2, by BD (2 mentions)
Cd3 clone 145 2c11, by Thermo Fisher Scientific (2 mentions)
Nk1.1 clone pk136, by BioLegend (2 mentions)
Cd4 clone rm4 4, by BD (2 mentions)
Fixable aqua dead cell staining, by Thermo Fisher Scientific (2 mentions)
Cd11b clone rm2817, by Thermo Fisher Scientific (2 mentions)
Cd127 clone a7r34, by BD (2 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (2 mentions)
Cd45 clone 30 f11, by Thermo Fisher Scientific (2 mentions)
Cd90.2 53 2 1, by BD (2 mentions)
Cd3ε clone 145 2c11, by BD (2 mentions)
Cd8b clone yts156, by BioLegend (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Gr1 clone rb6 8c5, by BD (2 mentions)
Cd3 clone 17a2, by BD (2 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (2 mentions)
Ly6g 1a8, by BioLegend (1 mentions)
Cd115 clone afs98, by Thermo Fisher Scientific (1 mentions)
15 protocols using cd19 clone 1d3
1
Multicolor Flow Cytometry Immunophenotyping
2
Flow Cytometric Analyses of B Cells and Tfh Cells
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For flow cytometric analyses of B cell populations, single cells were first prepared from lymph nodes by grinding with sieve. After staining with fluorescein isothiocyanate-labeled antibodies for CD19 (clone 1D3, Cat. 557398, BD Biosciences), cells were counted with BD LSRFortessa (BD Biosciences) and analyzed using BD FACSDiva software (BD Biosciences) and FlowJo software (FlowJo, Ashland, OR, USA). For flow cytometric analyses of Tfh cells, lymphocytes were stained with antibodies to CD4 (clone GK1.5, Cat. 552051, BD Biosciences), B220 (clone RA3-6B2, Cat. 562290, BD Biosciences), CD11b (clone M1/70, Cat. 563015, BD Biosciences), PD-1 (clone J43, Cat. 562584, BD Biosciences), CXCR5 (clone 2G8, Cat. 560615, BD Biosciences) and counted with BD LSRFortessa Cytometry (BD Biosciences).
3
Isolation and Characterization of Tumor-Infiltrating Lymphocytes
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In order to obtain tumor infiltrating lymphocytes, tumors from third passage B6CaP cells grown in parallel were mechanically disintegrated and digested for 1 h at 37°C in RPMI media with 10 mg/ml of Collagenase IV and 1 mg/ml of DNase. After digestion, white blood cells were isolated from tumors using anti CD45 antibodies conjugated with magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s protocol. Tumor infiltrating leukocytes were characterized with antibodies against CD3 (clone 145-2C11, eBioscience), CD19 (clone 1D3, BD Biosciences), CD49b (clone DX5, BD Biosciences), CD4 (clone H129.19, BD Biosciences), CD8 (clone 53-6.7, BD Biosciences), PD-1 (clone J43, eBioscience), Tim-3 (clone RMT3-23, eBioscience), Lag-3 (clone C9B7W, eBioscience) and CTLA4 (clone UC10-4F10-11, BD Biosciences). Flow through cells not captured by bead selection were stained with PDL-1 antibody (clone MIH5, eBioscience). For viability staining we used Live/Dead Aqua (Life Technologies). Gating controls were prepared with Fluorescence Minus One (FMO) stains of splenocytes or isotype control for tumor cells. Data were analyzed with FlowJo software (TreestarInc, Ashland, OR, USA).
4
Multiparametric Flow Cytometry of Lung Immune Cells
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Immune cells from BAL fluid or lung tissue cells were stained for 20 min at 4°C with Biotin rat anti‐mouse CD45 (clone 30‐F11; BD). Thereafter, cells were labelled for 20 min at 4°C with APC‐A750 Streptavidin (BD), APC rat anti‐mouse CD11c (clone N418; Thermo Fisher Scientific), APC‐A700 rat anti‐mouse CD11b (clone M1/70, Thermo Fisher Scientific), FITC rat anti‐mouse Ly6G (clone RB6‐8C5; Thermo Fisher Scientific), PE rat anti‐mouse Siglec F (clone E50‐2440, BD), PE‐CF594 rat anti‐mouse CD3 (clone 145‐2C11; BD), CD19 (clone 1D3, BD) and V500 IA/IE (clone M5/114.15.2; BD). The gating strategy is depicted in Figure S1 .
5
Comprehensive B Cell Immunophenotyping
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For flow cytometry, we used fluorochrome- or biotin-labeled antibodies against B220 (clone RA3–6B2), CD1d (clone 1B1), CD16/CD46 (Fc block; clone 2.4G2), CD19 (clone 1D3), CD21/CD35 (clone 7G6), CD23 (clone B3B4), CD35 (clone 8C12), CD38 (clone 90), CD95 (clone Jo2), CD138 (clone 281-2), GL-7 (clone GL-7), IgD (clone 11–26c.2a), IgM (clone R6–60.2), IgMa (clone DS-1), IgMb (clone AF6-78), and C3 split products (clone RmC11H9); all were purchased from BD Biosciences, Biolegend, eBioscience, or Cedarlane Laboratories. Rabbit polyclonal IgG anti-SRBC was prepared in-house (26 (link)). Staining was done in FACS buffer (PBS 2% FBS; Sigma Aldrich) or, when two or more Brilliant Violet™ antibody conjugates were used, in Brilliant Stain Buffer (BD Biosciences). All antibodies were used at ≤0.5 µg per 1x106 cells.
6
Immunohistochemistry and Flow Cytometry Protocol
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Five micron paraffin embedded tissue sections were processed for immuno-histochemistry as previously described (Sur et al., 2012b (link)). Rabbit polyclonal anti-Myc (Santa Cruz, sc-764; RRID:AB_631276 ) (1:500), Rabbit monoclonal anti Ki-67 (abcam, ab16667; RRID:AB_302459 ) (1:200), Goat polyclonal anti-Vimentin (Santa Cruz, sc-7557; RRID:AB_793998 ) (1:500), biotinylated goat anti-Rabbit IgG (Vector Laboratories, BA1000; RRID:AB_2313606 ) and biotinylated rabbit anti-Goat IgG (Vector Laboratories, BA5000; RRID:AB_2336126 ) (1:350) antibodies were used. For flow cytometry, single cell suspensions of spleen and bone-marrow and cells from peripheral blood were stained with Fc-block (CD16/CD32 clone 93, Biolegend, 101302, RRID:AB_312801 ) and subsequently with CD19 (clone 1D3, BD Biosciences, RRID:AB_11154223 ), TER119 (clone TER119, Biolegend 116210, RRID:AB_313711 ), CD3ε (clone 145–2 C11, Biolegend 100308, RRID:AB_312673 ), NK1.1(clone PK136, Biolegend, 108716, RRID:AB_493590 ), GR1/LY6G (clone RB6-8C5, Biolegend, 108410, RRID:AB_313375 ), CD4 (clone RM4-5, BD Biosciences, 563747) and CD8a (clone 53–6.7, BD Biosciences, 563332). Dead cells were visualized using Propidium iodide. Samples were analyzed using a BD LSRFortessa instrument.
7
Multiparametric Flow Cytometry Analysis
8
Multicolor Flow Cytometry Profiling
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Single-cell suspensions from spleen, peritoneal lavage, uterus, blood, inguinal (ILN) and para-aortic lymph nodes (PLN) were obtained and stained for cell surface markers for 30 min at 4 °C. The following anti-mouse fluorescently labeled antibodies were used: CD19 (clone 1D3) and CD4 (clone RM4-4; both from BD Biosciences, Germany), CD25 (clone 3C7), IgM (clone RMM-1), IgD (clone 11-26 c.2a) and B220 (clone RA3-6B2; all from Biolegend, San Diego, CA, USA). For detection of the intracellular expression of Foxp3, cell suspensions were fixed ON using Fix and Perm (ebioscience, Germany) and stained with anti-mouse Foxp3 (clone FJK-16s; ebioscience) for 30 min at 4 °C. Measurements were performed with a FACSCalibur and analyzed with CellQuestPro software (BD Biosciences).
9
Detecting Endogenous Gp100-Specific CD8+ T Cells
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For the detection of endogenous gp100-specific CD8+ T cells, we used an H-2Kb-KVPRNQDWL pentamer (ProImmune, Oxford, UK). Skin and draining LNs were analyzed for the presence of H-2Kb-KVPRNQDWL pentamer-positive CD8+ T cells. The cells were stained according to the manufacturer's protocol, and unspecific pentamer binding was excluded by pregating on viable CD19− (clone 1D3, BD Biosciences) NKp46− (clone 29A1.4, Biolegend) cells. For the detection of IFN-γ, we incubated cell suspensions from draining LNs with 1 μM gp100 peptide (KVPRNQDWL, Peptide&Elephants, Potsdam, Germany) for 48 h at 37 °C. IFN-γ release and antibody staining were performed as described above. For the calculation of gp100-responsive IFN-γ+ CD8+ cells, we used this formula: ((mean of IFN-γ+ CD8+ cells restimulated with gp100 peptide)−(mean of IFN-γ+ CD8+ cells cultured without gp100 peptide))/(mean of unstimulated pentamer+ CD8+ cells).
10
Multiparametric Immune Cell Profiling
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Lymphocytes were counted and subjected to viability staining (Fixable Aqua Dead Cell staining, Life Technologies, #L34957) and subsequently to receptor Fc blockade (BD #553142). Staining was performed using the following antibodies: CD4 (clone RM4-4, BD), CD45 (clone 30-F11 eBioscience), CD45.1 (clone A20, BD), CD45.2 (clone 104, BD) CD8a (clone 53-6.7, eBioscience or BD), CD8b (clone YTS156.7.7, BioLegend), CD11b (clone RM2817, Thermo Fisher), Ly6c (clone AL-21, BD), CD3ε (clone 145-2C11, BD), CD19 (clone 1D3, BD), CD90.2 (53-2.1, BD), CD127 (clone A7R34, BD), CX3CR1 (clone SA011F11, BioLegend), CXCR3 (clone CXCR3-173, Biolegend), CD103 (clone 2E7, eBioscience or Biolegend), CD69 (clone H1.2F3, BD), TCR-β (clone H57-597, BD, TCR-γδ (clone eBio-GL3, eBioscience). H2-Kb:SIINFEKL or H2-M3:fMIGWII tetramers were either produced in house or, for the former, obtained thorugh the NIH tetramer core. Samples were fixed (IC Fixation Buffer, eBioscience), washed, resuspended in FACS buffer and acquired with a LSRII flow cytometer (BD) either immediately or on the following day. For intracellular staining, the following antibodies were used: IFN-ϒ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) in Permeabilization buffer (eBioscience).
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