Cells were labelled with flow antibodies for 15 minutes in the dark in PBS containing 2% BSA. Labelled cells were washed twice and resuspended in PBS containing 2% BSA. The prepared samples were analyzed using an LSR-II flow cytometer (BD Bioscience) or sorted using an Aria II cell sorter (BD Bioscience). Anti-CD19-PeCy7 (561739), anti-CD3-PeCy7 (552774), anti-NK1.1-BV421 (562921), anti-NKp46-FITC (560756), anti-NKp46-AF647 (560755), anti-CD49b-PE (553858), anti-CD49a-PerCP-Cy5.5 (564862), anti-CD62L-APC (553152), anti-T-bet-APC (561264), anti-CD45.2-AF700 (560693), anti-CD45.2-FITC (553772), anti-Gata3-AF647 (560068), anti-RORγt-PE (562607), anti-CD127-V450 (561205), anti-CD117-PE (553869), anti-LPAM-1-APC (562376), anti-Flt3-BV421 (566292), anti-Ly-6A/E-APC (565355), and anti-CD122-PE (553362) were purchased from BD Bioscience. Anti-IFN-γ-AF-700 (505823) and anti-CD25-Pacific Blue (102022) were purchased from Biolegend. Anti-CD253-APC (17-5951-82), anti-Eomes-PE (12-4875-82), and anti-CD127-PerCP-Cy5.5 (45-1271-80) were purchased from eBioscience.
Anti cd49b pe
Manufactured by BD
Sourced in United States
Anti-CD49b-PE is a fluorescently labeled antibody that binds to the CD49b cell surface antigen. CD49b is a subunit of the very late antigen-4 (VLA-4) integrin complex, which plays a role in cell adhesion and migration. This product can be used in flow cytometry applications to identify and enumerate CD49b-positive cell populations.
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Lab products found in correlation
Facscalibur, by BD (2 mentions)
Anti cd4 apc, by BD (2 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Anti cd19 pe cy7, by BD (1 mentions)
Anti cd3 pe cy7, by BD (1 mentions)
Anti cd117 pe, by BD (1 mentions)
Anti cd62l apc, by BD (1 mentions)
Anti rorγt pe, by BD (1 mentions)
Aria 2 cell sorter, by BD (1 mentions)
Anti t bet apc, by BD (1 mentions)
Anti cd45.2 fitc, by BD (1 mentions)
Anti gata3 af647, by BD (1 mentions)
Anti cd122 pe, by BD (1 mentions)
Anti ifnγ af700, by BioLegend (1 mentions)
Anti cd25 pacific blue, by BioLegend (1 mentions)
Anti cd127 percp cy5, by Thermo Fisher Scientific (1 mentions)
Facs fortessa, by BD (1 mentions)
Anti cd19 fitc, by BD (1 mentions)
Anti cd8 pe cy7, by BD (1 mentions)
Anti ly6c pe, by BD (1 mentions)
6 protocols using anti cd49b pe
1
Multiparametric Flow Cytometry Analysis
2
Multicolor Flow Cytometry of Murine Immune Cells
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Splenocytes obtained from control or treated tumor bearing mice were liquid-nitrogen frozen and thawed before tests. Then, for lymphoid cell analysis, they were stained in a one-step test with the following fluorophore-labeled anti-mouse monoclonal antibodies (mAbs): anti-CD4-APC (BD Pharmingen, USA, RM4-5), anti-CD8-PE-Cy7 (BD Pharmingen, USA, 53–6.7), anti-CD49b-PE (BD Pharmingen, USA, DX5) and anti-CD19-FITC (BD Pharmingen, USA, 1D3). Phenotype analysis was carried out using the Becton Dickinson FACSCalibur apparatus with Cell Quest Software. For myeloid cell characteristics, the flow cytometry analysis of MDSC surface phenotype was performed as described previously [16 (link)] using fluorophore-labeled anti-mouse mAbs: anti-CD11b-PerCP-Cy5.5 (BD Pharmingen, USA, M1/70), anti-B220-APC (BD Pharmingen, USA, RA3-6B2), anti-Ly6G-APC-Cy7 (BD Pharmingen, USA, 1A8), anti-Ly6C-PE (BD Pharmingen, USA, AL-21) and anti-MHCII-FITC (BD Pharmingen, USA, 25-9-17). The cells were stained for 45 min at 4°C. The viability of spleen cells was assessed by incubation with DAPI dye. The analysis was carried out using Becton Dickinson FACSFortessa apparatus with FACSDiva software.
3
Multiparametric Flow Cytometry of Liver Immune Cells
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Flow cytometric analysis was performed on CytoFLEX (Beckman Coulter, CA, USA). To analyze liver infiltrating cells, hematopoietic cells were isolated by MACS Liver dissociation kit (Miltnyi Biotec Inc.) and Gentle MACS (Miltnyi Biotec Inc.). Antibodies were sourced from Biolegend: anti-CD3 phycoerythrin/Cyanine7 (PE/Cy7; 17A2), anti-CD4 phycoerythrin (PE; GK1.5), anti-B220 PE (RA3-6B2), anti-CD90.2 PE (30-H12), anti-CD11c PE/Cy7 (N418), anti-F4/80 fluorescein isothiocyanate (FITC; BM8), anti-NK1.1 Alexa Fluor 488 (PK136), anti-CD49b PE (DX5), anti-CD44 allophycocyanin (APC; IM7), anti-CD62L allophycocyanin/Cyanine7 (APC/Cy7 (MEL-14), anti-CD25 PE (PC61.5), anti-CD69 FITC (H1.2F3), anti-CD49d PE (MFR4.B), anti-CXCR3 APC (CXCR3-173), anti-PD-1 PE (29F.1A12), anti-KLRG-1 APC (2F1/KLRG1), anti-LFA-1 PE (H155-78), anti-LPAM-1 PE (DATK32), anti-H-2 Kb APC (AF6-88.5) and anti-CD8a FITC (53–6.7), anti-CD11b PE (M1/70) from BD Biosciences.
4
Immunophenotyping of Tumor-Infiltrating Lymphocytes
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Tumor cells obtained from control or treated mice were thawed, centrifuged, and stained in one-step test using monoclonal antibodies conjugated with fluorophores: anti-CD45 PE-Cy7, anti-CD4 APC, anti-CD8 FITC, and anti-CD49b PE (all from BD Biosciences). In order to eliminate dead cells during the analysis, cells were additionally stained with DAPI dye (Molecular Probes). The analysis was carried out using LSR Fortesssa with Diva software (Becton Dickinson). In order to determine the percentage of T regulatory lymphocytes in tumors, the cells were stained with monoclonal antibodies: anti-CD45 PE-Cy7 (BD Biosciences), anti-CD4 FITC (eBioscience), and anti-CD25 PE (eBioscience). Then, cells were fixed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to manufacturer instruction and incubated with anti-FoxP3 monoclonal antibody conjugated with APC (eBioscience). The analysis was carried out using LSR Fortessa with Diva software (Becton Dickinson).
5
Multiparameter Flow Cytometry Analysis
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For phenotypic analysis, single cell suspensions were stained as previously described 36 . For ex vivo Treg cell analysis, anti‐CD25‐PECy5, anti‐Helios‐FITC, anti‐CD8‐PE (from Biolegend, San Diego, CA, USA), anti‐CD4‐APC‐AF750 (from Thermo Fisher Scientific), and anti‐FoxP3‐APC (from eBiosciences, San Diego, CA, USA) were used. For in vitro induced Treg analysis, Zombie Aqua fixable dye, anti‐CD4‐APC and anti‐CD25‐PECy5 from Biolegend were used. For ex vivo DC analysis, cells were blocked with purified anti‐CD16/32, followed by staining with Zombie Aqua, anti‐I‐A/I‐E‐AF488, anti‐CD11c‐AF700, anti‐CD80‐PECy5 (from Biolegend), anti‐CD3‐PE, anti‐TER119‐PE, anti‐CD11b‐VF450, anti‐CD86‐APC, anti‐CD8‐PECy7 (from Tonbo Biosciences, San Diego, CA, USA), anti‐CD19‐PE, anti‐CD49b‐PE, streptavidin‐APCCy7 (from BD Biosciences, San Jose, CA, USA), anti‐CD103‐biotin and anti‐PD‐L1‐PerCP‐eFluor710 (from eBiosciences) were used.
For in vivo transfer experiments, anti‐CD45.1‐AF700, anti‐CD4‐FITC, anti‐CD25‐PECy5, streptavidin‐BV605 (from Biolegend), anti‐Vβ5‐biotin (from BD Biosciences), and anti‐FoxP3‐APC (from eBiosciences) were used for staining ovalbumin (OVA)‐specific T cells.
Samples were acquired in an Attune Acoustic Focusing Flow Cytometer (Thermo Fisher Scientific) and analyzed usingflowjo 10.0 software (Tree Star Inc., Ashland, OR, USA).
For in vivo transfer experiments, anti‐CD45.1‐AF700, anti‐CD4‐FITC, anti‐CD25‐PECy5, streptavidin‐BV605 (from Biolegend), anti‐Vβ5‐biotin (from BD Biosciences), and anti‐FoxP3‐APC (from eBiosciences) were used for staining ovalbumin (OVA)‐specific T cells.
Samples were acquired in an Attune Acoustic Focusing Flow Cytometer (Thermo Fisher Scientific) and analyzed using
6
Assaying Stem Cell-Like Phenotype
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Cells were seeded in 6-well plates and transfected with overexpression plasmids or siRNA as described above. Afterwards cells were trypsinized and cell pellets were suspended in propidium iodide (PI) buffer (0.2% Triton-X-100, 2 ng/mL Na-Citrate, and 0.1 mg/mL PI) and kept light-protected at 4°C for 1 hour.
Apoptosis (subG1 fraction) was analyzed using FACS Calibur (Becton Dickinson). For analysis of the stem cell-like phenotype cells were detached by scraping, washed with PBS containing 2% BSA, stained with anti-CD24-FITC, anti-CD44-PerCP-Cyc5.5 and anti-CD49b-PE (all from BD) and incubated at 4°C for 30 minutes. After incubation, cells were washed once with PBS containing 2% BSA and the CD24low/CD44high/CD49bhigh population was determined using FACS Calibur.
Apoptosis (subG1 fraction) was analyzed using FACS Calibur (Becton Dickinson). For analysis of the stem cell-like phenotype cells were detached by scraping, washed with PBS containing 2% BSA, stained with anti-CD24-FITC, anti-CD44-PerCP-Cyc5.5 and anti-CD49b-PE (all from BD) and incubated at 4°C for 30 minutes. After incubation, cells were washed once with PBS containing 2% BSA and the CD24low/CD44high/CD49bhigh population was determined using FACS Calibur.
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