Ascites cell pellets were incubated with 5 ml of red cell lysis buffer (0.17 M Tris-HCL, 0.16 M NH4Cl) for 5 min. Cells were spun down and resuspended in FACS buffer (1.5% heat inactivated FBS, 0.2% NaN3 in PBS). Equal numbers of viable cells were stained with PE-conjugated anti-CD11b, PE-Cy7-conjugated CD-11c, FITC-conjugated anti-Gr-1, and APC Cy7-conjugated anti-CD45 (all from eBiosciences). Flow-cytometric data were acquired on a BD FACSCanto II and analyzed using FACSDiva software (BD Biosciences). Viable cells were gated based on forward and side scatter profiles.
Fitc conjugated anti gr 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
FITC-conjugated anti-Gr-1 is a fluorescently labeled antibody that binds to the Gr-1 antigen, which is expressed on the surface of granulocytes, including neutrophils, eosinophils, and myeloid-derived suppressor cells. This antibody can be used in flow cytometry applications to identify and quantify these cell populations.
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Lab products found in correlation
Pe conjugated anti f4 80, by Thermo Fisher Scientific (2 mentions)
Pecy7 conjugated anti cd11b, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Pe conjugated anti cd11b, by Thermo Fisher Scientific (1 mentions)
Apc cy7 conjugated anti cd45, by Thermo Fisher Scientific (1 mentions)
Facs fortessa flow cytometer, by BD (1 mentions)
Apc conjugated anti cd4, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd19, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti cd3, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd11b, by Thermo Fisher Scientific (1 mentions)
Facsdiva 8, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Efluor450 conjugated anti cd45, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd3, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd3, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd8, by Thermo Fisher Scientific (1 mentions)
Percp conjugated anti cd4, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti pd 1, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti pd l1, by Thermo Fisher Scientific (1 mentions)
6 protocols using fitc conjugated anti gr 1
1
Multiparameter Flow Cytometry of Ascites Cells
2
Multiparametric Flow Cytometry Immunophenotyping
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Single cell suspensions (0.5 x 106 cells) were incubated for 15 min with Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences Pharmingen, San Diego, CA, USA), washed with PBS supplemented with 2% FCS and then stained for 30 min with the indicated conjugated antibodies. Cells were washed twice and fixed with 0.37% formaldehyde in PBS. FITC-conjugated anti-CD19, PE-conjugated anti-CD3, APC-conjugated anti-CD4, BV410-conjugated anti-CD8, APC-conjugated anti-CD11b, BV711-conjugated anti-CD11c, FITC-conjugated anti-Gr-1 and PE-conjugated anti-F4/80, were purchased from eBioscience (San Diego, CA, USA). Cells were analysed in a FACS Fortessa flow cytometer and data were processed with the FlowJo software (Becton Dickinson Labware, Franklin Lakes, NJ, USA).
3
Phenotyping tumor-infiltrating immune cells
4
MDSC Suppression of T Cell Proliferation
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Freshly isolated tumor cells from TC-1 tumors were stained with the following fluorophore-conjugated antibodies: PeCy7-conjugated anti-CD11b, FITC-conjugated anti-Gr1 and APC-conjugated anti-CD3 (eBioscience, catalog nr. 25-0112-82, 11-5931-82, 17-0031-82). Cells were washed and sorted on a FACS MoFloAstrios (Beckman Coulter) based on forward and sideward scatter. Cells were positively sorted into CD11b+Gr1+ MDSCs and negatively sorted into CD3+ T cells. As determined by flow cytometric re-analysis, the purity of the sorted MDSCs was >92%.
Spleens cells isolated from donor mice immunized twice with 5 × 106 SFVeE6,7 particles were cultured in 96-well round bottom plates for 7 d. On day 0 of culture, flow-sorted MDSCs, isolated from TC-1 tumors, were added at different ratios to these cultures. On day 4 of culture, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, catalog nr. C34554) for 10 min at 37oC. On day 5 of co-culture, recombinant IL-2 was added to the co-culture at a concentration of 5U/mL. As negative controls, splenocytes were cultured without stimulant. As positive controls, splenocytes were stimulated and cultured in the absence of MDSCs. At the end of the co-culture period, cells were collected and used for analysis.
Spleens cells isolated from donor mice immunized twice with 5 × 106 SFVeE6,7 particles were cultured in 96-well round bottom plates for 7 d. On day 0 of culture, flow-sorted MDSCs, isolated from TC-1 tumors, were added at different ratios to these cultures. On day 4 of culture, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, catalog nr. C34554) for 10 min at 37oC. On day 5 of co-culture, recombinant IL-2 was added to the co-culture at a concentration of 5U/mL. As negative controls, splenocytes were cultured without stimulant. As positive controls, splenocytes were stimulated and cultured in the absence of MDSCs. At the end of the co-culture period, cells were collected and used for analysis.
5
Flow Cytometry Analysis of Immune Cells
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For flow cytometry, the following antibodies for the analysis of human samples were used: FITC-conjugated anti-CD16, PE-conjugated anti-PD-L1, PerCP-conjugated anti- CD45, APC-conjugated anti-CD11b, APC-conjugated anti-CD14, FITC-conjugated anti-CD3, PE-conjugated anti-CD8, PerCP-conjugated anti-CD4 and APC-conjugated anti-PD-1. All human antibodies were purchased from BD Bioscience (San Jose, USA). Antibodies used in the mouse experiments were purchased from eBioscience (San Diego, CA) as follows: FITC-conjugated anti-Gr-1, PE-conjugated anti-PD-L1, PerCP-conjugated anti-CD11b, APC-conjugated anti-CD48, APC-conjugated anti-CD8, FITC-conjugated anti-CD3, PE-conjugated anti-PD-1, and PerCP-conjugated anti-CD4. Data were analysed with FlowJo5.6.7 (Tree star, Inc., Ashland, OR, USA).
6
Tumor Immune Profile Characterization
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Tumor masses were minced and digested with a mixture of 0.3 mg/ml DNase I (Sigma-Aldrich) and 0.25 mg/ml Liberase TL (Roche) in serum-free RPMI at 37°C for 25 min. Then the digested pieces were gently pressed between the frosted edges of two sterile glass slides, and the cell suspension was filtered through a 40 μm cell strainer (BD Biosciences). The immune subsets in the tumors were determined by flow cytometry and data were acquired using a FACS flow cytometer (BD Biosciences, San Jose, CA). For intracellular IFN-γ staining, harvested cells were stimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 4 h and incubated for the last 1 h with brefeldin A (10 μg/ml). Cells were subjected to intracellular cytokine analysis with anti-IFN-γ antibody. The antibodies used for FACS include APC-conjugated anti-CD45, Pacific blue-conjugated anti-CD8, FITC-conjugated anti-IFN-γ, Alex Flour 488-conjugated anti-p-S6, PerCP/Cy5.5-conjugated anti-CD4, PE-conjugated anti-NK1.1, FITC-conjugated anti-TCRγδ, PE-Cy7-conjugated anti-CD11b, FITC-conjugated anti-Gr-1, and PE-conjugated anti-MHC-II (eBioscience). Data were analyzed by FlowJo software (TreeStar Inc., San Carlos, CA).
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