Assessment of apoptosis was measured by flow cytometry using the active caspase-3 as previously reported (25 (link), 26 (link)). To discriminate viable from non-viable, cells were stained and incubated in FVS V500 dye (BD Bioscience, US) for 15 min at room temperature. For surface staining, 1 × 106 cells were labeled with appropriate antibodies: MHCII (M5/114.15.2), CD11b (M1/70) and F4/80 (PM8) purchased from BD Biosciences (Fanklin Lakes, NJ, US) and eBioscience (San Diego, CA, US). For intracellular staining, cells were washed, fixed and permeabilized with 1X Fixation-Permeabilization buffer and then stained as per manufacturer's instruction with appropriate antibodies: caspase-3 (C92-605) and Bcl-2 (RUO) (all from BD Biosciences, US). After the incubation period cells were resuspended in FACS buffer for acquisition. Acquisition was performed using BD LSRFortessa (BD Biosciences), and data were analyzed using FlowJo software (Treestar, Ashland, OR, US).
Mhc 2 m5 114.15.2
Manufactured by BD
Sourced in United States
MHC II (M5/114.15.2) is a monoclonal antibody that recognizes the major histocompatibility complex class II (MHC II) proteins. MHC II molecules are cell surface receptors that present peptide antigens to T cells, initiating an immune response. This antibody is a widely used tool in immunology research.
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Lab products found in correlation
Cd11b m1 70, by BD (3 mentions)
Cd45 30 f11, by BioLegend (3 mentions)
Lsrfortessa, by BD (2 mentions)
F4 80 bm8, by BioLegend (2 mentions)
Cd4 rm4 5, by BioLegend (2 mentions)
Granzyme b, by BioLegend (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Ly6c al 21, by BD (2 mentions)
Ly6g 1a8, by BioLegend (2 mentions)
Cd8a 53 6, by BD (1 mentions)
Cd11c n418, by BioLegend (1 mentions)
Ter119, by Thermo Fisher Scientific (1 mentions)
Cd3e 145 2c11, by BD (1 mentions)
Cd16 cd32 93, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by FlowJo (1 mentions)
Dnase 1, by Roche (1 mentions)
Collagenase d, by Roche (1 mentions)
B220 ra3 6b2, by BioLegend (1 mentions)
Gr 1 rb6 8c5, by BioLegend (1 mentions)
Cd69 h1.2f3, by BioLegend (1 mentions)
8 protocols using mhc 2 m5 114.15.2
1
Apoptosis Assessment by Flow Cytometry
2
Flow Cytometric Analysis of Migratory DCs
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C57BL/6J mice were vaccinated intradermally at the right flank with either Alexa Fluor 647-labeled OVA (OVA-647, 10 µg) alone or OVA-647 (10 µg) + heat-iMVA (an equivalent of 107 pfu) in a volume 100 µl PBS. Skin draining lymph nodes (dLNs) at the right inguinal area were harvested at 24 h post injection, digested with Collagenase D (400 U/ml, Roche Diagnostics) and DNase I (50 μg/ml, Roche Diagnostics), and analyzed by flow cytometry for OVA-647 intensities and CD86 expression of the migratory DC and resident DC populations in the skin dLNs. The antigens and clone designations for the antibodies are as follows: BioLegend: CD11c (N418, cat# 117320), CD11b (M1/70, cat# 101226), MHC-II (M5/114.15.2, cat# 107645), CD3e (145-2C11, cat# 100341), CD8a (53-6.7, cat# 140418); BD Biosciences: CD19 (1D3, cat# 562701), CD49b (DX5, cat# 563063), Thermo Fisher: CD16/CD32 (93, cat# 13-0161-82), CD103 (2E7, cat# 11-1031-82), CD207 (eBioL31, cat# 12-2075-82), and TER-119 (TER-119, cat# 48-5921-82). All antibodies were used at 1:200. Cells were analyzed on the BD LSR Fortessa or LSR II flow cytometer and data were analyzed with FlowJo software (version 10.5.3).
3
Tumor-Infiltrating Immune Cell Analysis
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Twenty-four hours after the last exercise session, mice were anesthetized, and tumors were harvested, and samples were processed as previously described (3 (link)). Flow cytometry data were obtained using an LSRII flow cytometer (BD Biosciences) and/or Aurora (Cytek) and analyzed with FlowJo software. The double/aggregated cells were gated out using forward scatter area (FSC-A) versus forward scatter width (FSC-W) and side scatter area (SSC-A) versus side scatter width (SSC-W). Different fluorophores conjugated with the following mAb were used: CD45 (30-F11, Biolegend), TCRβ chain (H57-597, Biolegend), CD4 (RM4-5, Biolegend), FOXP3 (150D, Biolegend), CD8a (53-6.7, Biolegend), CD11c (N418, Biolegend), Gr1 (RB6-8C5, Biolegend), MHC-II (M5/114.15.2, BD Biosciences) CD11b (M1/70, Biolegend), B220 (RA3-6B2, Biolegend), F4/80 (BM8, Biolegend), IFNγ (XMG1.2, Biolegend), Granzyme B (GB11, Biolegend), Ki67 (16A8, Biolegend), CD62L (W18021D, Biolegend), CD44 (NIM-R8, Biolegend), CD69 (H1.2F3, Biolegend), CXCR3 (173, Biolegend), IL15Rα (6B4C88, Biolegend), IL6Rα (D7715A7, Biolegend), CD247 (Biolegend, 10F.9G2).
4
Comprehensive Murine Lung Cell Analysis
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Whole lungs were removed and chopped with razor blades, incubated with type IV collagenase (Worthington) at 37 °C for 40 min, then homogenized through a 70-µm cell strainer (Falcon). Remaining red blood cells were lysed using 1× red blood cell lysis buffer (BD Biosciences). Cells were stained with Fixable Viability Dye eFluor® 455 (eBioscience). Anti-mouse immunophenotyping antibodies were diluted in FACS buffer (3% FBS, 2 mM EDTA, 1× PBS) to 5 µg per mL along with Fc block (anti-mouse CD16/CD32; 5 µg per mL, BD), and cells were stained for 30 min on ice in three groups (AMφ: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), and Siglec-F (E50-2440; BD); monocyte and neutrophil: Ly-6C (AL-21; BD), Ly-6G (1A8; BD), and CD11b (M1/70; BD); dendritic cell: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), MHC II (M5/114.15.2; BD), and CD103 (M290; BD); NK cells: CD3 (17A2; BD), NK-1.1 (PK136; BD), and CD49b (DX5; BD)). After the staining, cells were washed twice with FACS buffer and then fixed in 2% para-formaldehyde in FACS buffer for 15 min. Cell numbers were counted using AccuCount Fluorescent Particles (Spherotech). All data were collected on an LSR II flow cytometer (BD) and analyzed using FlowJo software.
5
Comprehensive Immunophenotyping and Signaling Analysis
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For IHC, antibodies used were from Cell Signaling Technology for CD31 (#77699S) and for CD8 (#98941S). For immunoprecipitation performed for Western blot analysis, antibodies used were from R&D Systems [Goat-anti-mouse antibody, VEGFR2 (#AF644) and AXL (#AF154)]. For Western blot analysis, antibodies used were from Cell Signaling Technology [VEGFR2 (#9698), pAXL (#5724), and cMET (#4560S)], Thermo Fisher Scientific [pVEGFR2 (#44-1047G) and pMET (#44-88G)] and Abcam [AXL (#ab215205)].
For flow cytometry, antibodies used for surface staining were: PD-L1 (MIH5; Thermo Fisher Scientific), PD-1 (29F.1A12; BioLegend), CD3 (145-2C11; BioLegend), CD11b (M1/70; BioLegend), CD45 (30-F11; BioLegend), CD8 (53-6.7; BioLegend), NKp46 (29A1.4; BioLegend), CD4 (RM4-5; BioLegend), Ly6G (1A8; BioLegend), CD11c (N418; Thermo Fisher Scientific), F4/80 (T45-2342; BD Biosciences), Ly6C (AL-21; BD Biosciences), MHC II (M5/114.15.2; BD Biosciences), CD19 (6D5; BioLegend), and CD25 (PC61; BioLegend). For intracellular staining, the antibodies used were: Arginase 1 (A1exF5; Thermo Fisher Scientific), Foxp3 (FJK-16s; eBioscience), Ki67 (Sola15; eBioscience), IFN (XMG1.2; BioLegend), and granzyme B (GB11; BioLegend).
For flow cytometry, antibodies used for surface staining were: PD-L1 (MIH5; Thermo Fisher Scientific), PD-1 (29F.1A12; BioLegend), CD3 (145-2C11; BioLegend), CD11b (M1/70; BioLegend), CD45 (30-F11; BioLegend), CD8 (53-6.7; BioLegend), NKp46 (29A1.4; BioLegend), CD4 (RM4-5; BioLegend), Ly6G (1A8; BioLegend), CD11c (N418; Thermo Fisher Scientific), F4/80 (T45-2342; BD Biosciences), Ly6C (AL-21; BD Biosciences), MHC II (M5/114.15.2; BD Biosciences), CD19 (6D5; BioLegend), and CD25 (PC61; BioLegend). For intracellular staining, the antibodies used were: Arginase 1 (A1exF5; Thermo Fisher Scientific), Foxp3 (FJK-16s; eBioscience), Ki67 (Sola15; eBioscience), IFN (XMG1.2; BioLegend), and granzyme B (GB11; BioLegend).
6
Multicolor Flow Cytometry of Immune Cells
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Dead cells were stained using LIVE/DEAD® fixable dead cell stain kit (Invitrogen) as described by the manufacturer. Fluorochrome-coupled antibodies recognizing mouse CD3 (clone 145-2C11), CD4 (RM4-5), CD11b (M1/70), CD11c (N418), CD19 (MB19-1), CD25 (PC61.5), CD44 (IM7), CD49b (DX5), CD62L (MEL-14), CD86 (GL1), CD90.2 (53-2.1), iNOS (C-11), and MHCII (M5/114.15.2) were purchased from either BD Biosciences, BioLegend, eBioscience or Santa Cruz Biotechnology. Specific antibody staining was performed at 4 °C in the dark for 15 min (surface staining) or 30 min (intracellular staining using Foxp3/Transcription Factor Staining Buffer Set from eBioscience). Spleens were stained in a total volume of 100 μl, and lung samples in 200 μl. To reduce unspecific antibody binding, surface and intracellular staining were performed in the presence of anti-CD16/CD32 (2.4G2, BioXCell) antibodies and ChromPure rat IgG whole molecule (Jackson ImmunoResearch), respectively, and using predetermined optimal antibody dilutions. Carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) staining (10 μM) was performed at 2 × 107 cells/ml of PBS for 2 min and 45 s at room temperature. Reaction was stopped by adding a tenfold volume of complete RPMI 1640 medium (Invitrogen) containing 10% fetal calf serum (cRPMI) followed by two washing steps.
7
Multiparametric Immune Cell Profiling
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Isolated cells were washed in FACS staining buffer and incubated with anti-CD16/32 (clone 2.4G2) to block FcRs for 10 mins followed by an incubation with Live/Dead fixable yellow dead cell stain (ThermoFisher) and fluorescent-conjugated monoclonal antibodies (mAbs) for 30 min.
The following anti-mouse mAbs were used for analysis: CD45 (30-F11; BioLegend), CD3 (17A2; BioLegend), F4/80 (BM8; BioLegend), CD11b (M1/70; BioLegend), Ly6C (HK1.4; BioLegend), Ly6G (1A8; BioLegend), CD335 (29A1.4; BioLegend), MHC II (M5/114.15.2; BD Biosciences), CD206 (C068C2; BioLegend). Depending on the experiment, samples were either immediately analyzed by flow cytometry using a Sony SP6800 Spectral Analyzer or further processed to determine Perforin-2 mRNA levels. Branched oligonucleotide signal amplification was used to determine Perforin-2 mRNA levels in individual cells (PrimeFlow; ThermoFisher Scientific).
Briefly, single cell suspensions were stained for surface antigens as described above, then fixed, permeabilized, and incubated with probes specific for Perforin-2 transcripts (Assay Id: VB1-20172-PF; ThermoFisher Scientific). Cells were then subjected to a series of signal amplification cycles and then analyzed by flow cytometry as described above using FlowJo software (BD Biosciences).
The following anti-mouse mAbs were used for analysis: CD45 (30-F11; BioLegend), CD3 (17A2; BioLegend), F4/80 (BM8; BioLegend), CD11b (M1/70; BioLegend), Ly6C (HK1.4; BioLegend), Ly6G (1A8; BioLegend), CD335 (29A1.4; BioLegend), MHC II (M5/114.15.2; BD Biosciences), CD206 (C068C2; BioLegend). Depending on the experiment, samples were either immediately analyzed by flow cytometry using a Sony SP6800 Spectral Analyzer or further processed to determine Perforin-2 mRNA levels. Branched oligonucleotide signal amplification was used to determine Perforin-2 mRNA levels in individual cells (PrimeFlow; ThermoFisher Scientific).
Briefly, single cell suspensions were stained for surface antigens as described above, then fixed, permeabilized, and incubated with probes specific for Perforin-2 transcripts (Assay Id: VB1-20172-PF; ThermoFisher Scientific). Cells were then subjected to a series of signal amplification cycles and then analyzed by flow cytometry as described above using FlowJo software (BD Biosciences).
8
Multiparameter Flow Cytometry of Murine and Human Myeloid Cells
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Murine cells were stained using standard protocols for the surface markers CD45 (clone 30-F11), CD11b (M1/70, eBioscience), Ly6C (HK1.4, eBioscience), Ly6G (RB6-8C5, eBioscience), F4-80, (BM8, eBioscience), MHCII (M5/114.15.2, BD), CD86 (GL1, eBioscience), CD4 (GK1.5, eBioscience), CD8 (53-6.7, eBioscience), CD25 (PC61.5, eBioscience), and LIVE/DEAD Fixable Near-IR Dead Cell Stain. Intracellular staining for Arginase 1 (R&D) (polyclonal sheep IgG), iNOS (CXNFT, eBioscience), TNFa (MP6-XT22, eBioscience) was performed on cells fixed/permeabilized with the Transcription Factor Staining Buffer Set (eBioscience). Human MDSCs were stained for the surface markers CD11b (ICRF44, eBioscience), CD33 (WM-53, eBioscience), HLA-DR (L234, BioLegend), CD4 (OKT4, eBioscience), CD8 (HIT8A, eBioscience). Cytokine production was assessed after 3 hours of PMA + Ionomycin stimulation. Cells were analyzed on the BD Symphony and sorted on the BD FACS Aria.
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