Tumor cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described by Rossowska et al. (30 (link)). Briefly, tumor-derived cells were stained with LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher) and then stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 AlexaFluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD86 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE, anti-CD44 PE-Cy7, anti-CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using the FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).
Anti cd3 bv650
Manufactured by BioLegend
Sourced in Germany
Anti-CD3-BV650 is a fluorescently-labeled monoclonal antibody that binds to the CD3 antigen expressed on the surface of T cells. It is designed for use in flow cytometry applications to identify and quantify T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4 fitc, by BioLegend (3 mentions)
Anti cd206 apc, by BioLegend (3 mentions)
Anti cd11c pe cy7, by BioLegend (3 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (2 mentions)
Diva software, by BD (2 mentions)
Anti ly6g apc cy7, by BioLegend (2 mentions)
Live dead fixable violet dead staining kit, by Thermo Fisher Scientific (2 mentions)
Facs fortessa flow cytometer, by BD (2 mentions)
Anti cd19 pe cf594, by BD (2 mentions)
Anti cd49b pe cf594, by BD (2 mentions)
Anti cd45 bv605, by BD (2 mentions)
Anti cd11b percp cy5, by BioLegend (2 mentions)
Anti cd11c bv650, by BioLegend (2 mentions)
Anti f4 80 alexafluor 700, by BioLegend (2 mentions)
Anti mhc 2 fitc, by BioLegend (2 mentions)
Anti cd45 bv605, by BioLegend (2 mentions)
Anti cd8 apc fire 750, by BioLegend (2 mentions)
Anti cd3 pe cf594, by BD (2 mentions)
Anti cd25 pe, by BioLegend (2 mentions)
Anti ly6c pe, by BioLegend (2 mentions)
6 protocols using anti cd3 bv650
1
Identification of Tumor-Infiltrating Immune Cells
2
Comprehensive Immune Profiling of Tumor and Spleen Cells
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Tumor cells and spleen cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described previously (46 (link)). Briefly, tumor single-cell suspensions were stained with the LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher Scientific, Inc.) and then labelled with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 Alexa Fluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD80 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE (all from BioLegend) for lymphocyte identification. Then, the cells were fixed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. In spleen single cell suspension only the lymphocyte identification was performed according to the procedure described above. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).
3
Multiparameter Profiling of Immune Cells
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THP-1 macrophages and bone marrow-derived macrophages were detached using ice-cold PBS, 0.1% EDTA. Lamina propria cells, peritoneal immune cells and lung immune cells were isolated as described previously.13 (link),44 (link),45 The cells were then incubated with FcR blocking antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min and stained with anti-CD45-Pacific Blue, anti-CD3-BV650, anti-NK1.1-BV650, anti-B220-BV650, anti-CD11b-BV605, anti-CD11c-PECy7, anti-Ly6C-PerCPCy5.5, anti-F4/80-Fitc, anti-CD64-PE, anti-MHC-II-AF700, anti-CD206-PE-TexasRed, anti-pSTAT6-APC (all from BioLegend, mouse cells) anti-CD124-PE, anti-CD206-APC, (all from Biolegend, human cells) for 15–30 min. ZOMBI-NIR live dead stain (BioLegend, San Diego, CA) was used for discrimination between live and dead cells in all experiments. For pSTAT6 staining, cells were permeabilized using the FoxP3 staining kit from eBioscience. Samples were acquired on an LSRII cytometer (BD, Franklin Lakes, NJ), and analyzed using FlowJo (Tree Star, Inc. Ashland, OR). For RNA expression analysis of lung immune cell susbsets, lung homogenates were sorted on a MoFlo Asterios EQ cell sorter (Beckman Coulter Life Sciences, Krefeld, Germany).
4
Multicolor Flow Cytometry Immunophenotyping
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The cells were blocked with 1 µg anti-CD16/32 per 106 cells for 15 min at 4 °C. Cells were incubated with antibodies in the dark for 1 h on ice. After washing with 1% BSA in PBS, cells were filtered and resuspended in the cell staining buffer for characterization of murine immune cell subsets according to a previously reported protocol [31 ]. The following antibodies were used: anti-CD3-BV650, anti-CD11b-Pacific Blue, anti-CD11c-PE/Cy7, anti-CD19-BV605, anti-CD45-Per-CP-Cy5.5, anti-MHC-II-BV510, anti-F4/80-PE, and anti-Ly6G-FITC (BioLegend, CA, USA). Data acquisition was performed on a CytoFLEX Flow Cytometer (Beckman Coulter, CA, USA) and analyzed with Kaluza software (version 4.2, Beckman Coulter).
5
Apoptosis and Tumor-Infiltrating Lymphocyte Analysis
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To assess apoptosis, the cells were cultured for 24 h in RIPM 1640 medium containing normal glucose (2 g/L) or low glucose (40 mg/L) levels. The cells were centrifuged and resuspended in 0.5 ml annexin V-binding buffer (KeyGEN Biotech, China). Thereafter, 5 μl annexin V-APC and 7-AAD were added to the samples and incubated at RT for 10 min in the dark. The samples were then analyzed on a FACS Caliber flow cytometer (BD Biosciences, US).
The lymphocytes from 4T-1-injected BALB/c mice were isolated as follows: the tumor tissues from the mice were sectioned and digested with 2 mg/ml collagenase IV and 100 ng/ml DNase I (sigma) at 37 °C for 30 min. The tissues were then added to RPMI 1640 media supplemented with 10% FBS and 0.5 mM EDTA and separated by discontinuous 30–70% Percoll (GE Healthcare). After stimulation with PMA/Ionomycin and BFA (sigma) for 6 h at 37 °C, the cells were harvested for surface staining and intracellular staining (BD Pharmingen), according to the manufacturer’s instructions. The antibodies were anti-CD45-BV510, anti-CD45-APCcy7, anti-CD3-APC, anti-CD3-BV650, anti-CD4-FITC, anti-CD4-PEcy7, anti-PD-1-BV785, anti-PD-1-PE, anti-TIGIT-BV421, anti-TCR α/β-PE anti-IFN-γ-BV785, anti-IFN-γ-APC, anti-TNF-α-BV421 and anti-TNF-α-APC (BioLegend). All the data were collected by BD FACS Celesta flow cytometry and processed by Flow Jo software.
The lymphocytes from 4T-1-injected BALB/c mice were isolated as follows: the tumor tissues from the mice were sectioned and digested with 2 mg/ml collagenase IV and 100 ng/ml DNase I (sigma) at 37 °C for 30 min. The tissues were then added to RPMI 1640 media supplemented with 10% FBS and 0.5 mM EDTA and separated by discontinuous 30–70% Percoll (GE Healthcare). After stimulation with PMA/Ionomycin and BFA (sigma) for 6 h at 37 °C, the cells were harvested for surface staining and intracellular staining (BD Pharmingen), according to the manufacturer’s instructions. The antibodies were anti-CD45-BV510, anti-CD45-APCcy7, anti-CD3-APC, anti-CD3-BV650, anti-CD4-FITC, anti-CD4-PEcy7, anti-PD-1-BV785, anti-PD-1-PE, anti-TIGIT-BV421, anti-TCR α/β-PE anti-IFN-γ-BV785, anti-IFN-γ-APC, anti-TNF-α-BV421 and anti-TNF-α-APC (BioLegend). All the data were collected by BD FACS Celesta flow cytometry and processed by Flow Jo software.
6
Comprehensive Flow Cytometry Analysis of Immune Cells
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For flow cytometry of immune cells, lamina propria immune cells were isolated as described.83 (link) For analysis of myeloid immune cells, the cells were washed in PBS, incubated with FcR blocking antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 minutes, and stained with anti-CD45–Pacific Blue, anti-CD3–BV650, anti-NK1.1–BV650, anti-B220–BV650, anti-CD11b–BV605, anti-CD11c–PECy7, anti-Ly6C–PerCPCy5.5, anti-F4/80–APC, anti-CD64–PE, and anti–MHC-II–AF700 (all from BioLegend, San Diego, CA) for 15–30 minutes. ZOMBI-NIR live dead stain (BioLegend) was used for discrimination between live and dead cells. For cytokine staining, the cells were incubated with ionomycin and PMA in the presence of Brefeldin A for 3.5 hours before surface staining with anti-CD25–AlexaFluor700, anti-CD3–PerCPCy5.5, anti-CD4–BV510, and anti-CD8–BV570 for 15 minutes. Cells then were fixed with the FoxP3 staining kit (eBioscience) according to the manufacturer’s instructions, stained with anti-FoxP3–Pacific Blue, anti–IFN-γ–PECy7, anti-IL17–APC, anti-TNFα–BV650, and anti-IL22–PE for 30 minutes, washed in PermWash buffer (eBioscience), samples were acquired on an LSRII cytometer (BD, Franklin Lakes, NJ), and analyzed using FlowJo (Tree Star, Inc, Ashland, OR). Gating strategy for T and myeloid cells was performed as described previously.32
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