Tumor tissue was removed and cut into small fragments. Single tumor cell suspension was obtained by digestion with tumor disassociation kit (Miltenyi Biotec, USA) at 37 °C and then filtering through 70 µm cell strainers. Mononuclear cells were enriched by a two-level percoll gradient. Following a wash with PBS, cells were stained with Aqua Live/Dead stain and fluoro-conjugated antibodies specific to cell surface markers (BioLegend). After fixation-permeabilization, cells were stained with fluorochrome labeled antibodies specific to intracellular markers (BioLegend) or with the isotype controls. BD LSRFortessa was sued for cell acquisition and data analysis was carried out using the FlowJo software.
Tumor disassociation kit
Manufactured by Miltenyi Biotec
Sourced in United States
The Tumor Dissociation Kit is a laboratory equipment designed for the mechanical and enzymatic dissociation of solid tumor tissue samples. It provides the necessary components to obtain single-cell suspensions from tumor samples for further analysis or cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Anti cd4 fitc, by BD (3 mentions)
Lsrfortessa, by BD (2 mentions)
Golgi stop and monesin, by Thermo Fisher Scientific (2 mentions)
Anti cd8 percp, by BD (2 mentions)
Anti ly6c apc, by BD (2 mentions)
Anti ifnγ pe cf594, by BD (2 mentions)
Anti ly6g af700, by BD (2 mentions)
Live dead dye, by BD (2 mentions)
Anti cd8 apc cy7, by BD (1 mentions)
Anti ifn γ apc, by BioLegend (1 mentions)
Anti cd3 percp, by BioLegend (1 mentions)
Anti pd 1 pe, by BioLegend (1 mentions)
Anti pd l1 pe cy7, by BioLegend (1 mentions)
Anti cd62l apc, by BioLegend (1 mentions)
Anti cd44 apc cy7, by BioLegend (1 mentions)
Anti granzyme b fitc, by BioLegend (1 mentions)
Anti cd4 pecf594, by BD (1 mentions)
Anti mhc 1 pe, by BioLegend (1 mentions)
Anti cd25 percp, by BioLegend (1 mentions)
7 protocols using tumor disassociation kit
1
Single-Cell Analysis of Tumor Tissue
2
Comprehensive Immune Phenotyping of Tumor Samples
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Tumor tissue was harvested and cut into small fragments followed by digestion with a tumor disassociation kit (Miltenyi Biotec, USA) for 30 min, and then filtered by 70 μm cell strainers. Cells were stimulated with phorbol myristate acetate (PMA)/ionomyocin in the presence of Golgi stop and Monesin (eBioscience) for 4 h. Cells were washed with PBS and then stained with Live/Dead dye and fluorochrome-labeled antibodies specific to cell surface markers [anti-CD45 Alexa 700 (Clone 30-F11, BioLegend), anti-PD-L1 PE-Cy7 (Clone 10F.9G2, BioLegend), anti-CD3 PerCP (Clone 45-2C11, BioLegend), anti-CD8 APC-Cy7 (Clone 53–6.7, BD), anti-CD11c FITC (ab210308, Abcam), anti-PD1 PE (Clone RMP1–30, BioLegend), anti-CD4 PE-CF594 (Clone RM4–5, BD), anti-MHC-I PE (Clone 28–14-8, BioLegend), anti-CD44 APC-Cy7 (Clone IM7, BioLegend), anti-CD62L APC (Clone MEL-14, BioLegend), anti-CD25 PerCP (Clone 3C7, BioLegend), anti-CD127 FITC (Clone A7R34, BioLegend), anti-ICOS PE-Cy7 (Clone 7E.17G9, BioLegend)]. After fixation–permeabilization, cells were stained with fluoro-conjugated antibodies specific to intracellular markers [anti-Ki67 APC (Clone 16A8, BioLegend), anti-IFNγ APC (Clone XMG1.2, BioLegend), anti-granzyme B FITC (Clone GB11, BioLegend)] or with the isotype control. Flow cytometry analysis was performed on BD LSRFortessa and data were analyzed by FlowJo V10.
3
Tumor-infiltrating T cell isolation
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Single-cell suspensions from tumors were prepared using the Tumor Disassociation Kit, mouse, and GentleMACS (Miltenyi Biotec) according to the manufacturer’s procedures. Living lymphocytes were isolated followed by density gradient centrifugation on a 70%/40% Percoll (GE Healthcare, Chicago, IL, USA) gradient. After density gradient centrifugation, T cells were isolated with CD90.2 MicroBeads and cultured overnight in culture medium. The cultured T cells were stained with SART293–101 tetramer, anti-CD8-APC mAb (clone KT15), and anti-CD90.2-BV510 mAb. CD90.2+CD8+ cells were analyzed by FACSVerse (BD Bioscience, San Jose, CA, USA).
4
Phenotyping Myeloid-Derived Suppressor Cells
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M-MDSC and G-MDSC were phenotyped using flow cytometry. Tumor was cut into small fragments followed by digestion with tumor disassociation kit for 30 min (Miltenyi Biotec, USA), and then filtered by 70 µm cell strainers. Mononuclear cells was enriched by subjecting the single cell suspension to percoll gradient. Briefly, 70% percoll (4 ml) was added to the centrifuge tube, 30% percoll (4 ml) was carefully layered, and then tumor suspension (4 ml) was carefully layered on the top. After centrifuging at 1500 rpm for 15 mins, the buffy coat layer in the middle was collected and washed with PBS, followed by staining with anti-CD4 FITC, anti-CD8 Percp, anti-Ly6G AF700, anti-Ly6C APC, anti-CD11b APC-Cy7, anti-IFNγ PE-CF594, and anti-granzyme B PE antibodies, along with appropriate isotype controls (all from BD) for flow cytometry analysis (BD FACSCalibur). M-MDSC were defined as CD11b+Ly6C+Ly6G- and G-MDSC were defined as CD11b+Ly6G+Ly6C-.
5
Tumor-Infiltrating Immune Cell Profiling
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Tumor tissue was harvested and cut into small fragments followed by digestion with tumor disassociation kit for 30 min (Miltenyi Biotec, USA), and then filtered by 70 µm cell strainers. Mononuclear cells were enriched by percoll gradient centrifuging of the single cell suspension. Cells were washed with PBS and stained with Live/Dead dye, anti-CD4 FITC, anti-CD8 PE-Cy7, anti-Ki67 APC, anti-IFNγ PE-CF594, anti-FoxP3 Percp and anti-granzyme B PE antibodies, along with appropriate isotype controls (all from BD) for flow cytometry analysis (BD FACSCalibur).
6
Phenotyping and Sorting of G-MDSC and M-MDSC
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G-MDSC and M-MDSC were phenotyped using flow cytometry. Tumor was cut into small fragments followed by digestion for 30 min with tumor disassociation kit (Miltenyi Biotec, USA), and then filtered by 70 µm cell strainers. The percoll gradient was used to enrich mononuclear cells. After washing with PBS, cells were stimulated with PMA/ionomyocin in the presence of Golgi stop and Monesin (eBioscience, San Diego, CA, USA) for 4 h. Cells were washed with PBS and then stained with Live/Dead dye, anti-CD11b PE-Cy7, anti-Ly6G AF700, anti-Ly6C APC, anti-CD4 FITC and anti-CD8 Percp antibodies (all from BD). After fixation-permeabilization, cells were stained with anti-IFNγ PE-CF594 antibody, along with appropriate isotype controls (all from BD) for flow cytometry analysis (BD FACSCalibur). G-MDSC was defined as CD11b+Ly6G+Ly6C- and M-MDSC was defined as CD11b+Ly6C+Ly6G-. MDSC subsets were sorted with corresponding beads from the tumor lysates for western blot assay.
7
Tumor-Infiltrating Lymphocyte Isolation
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Blood was collected from normal mice and tumor-bearing mice. Tumor tissues were removed from the mice after euthanasia. Single tumor cell suspension was obtained by slicing of the tumor tissues followed by digestion with tumor disassociation kit (Miltenyi Biotec, USA) at 37 °C. After digestion, the suspension was filtered through 70 µm cell strainers to get rid of debris. Mononuclear cells were enriched by percoll gradient centrifuging. Cells were stimulated with PMA/ionomyocin in the presence of Golgi stop and Monesin (eBioscience) for 4 h. Cells were washed with PBS and then stained with Live/Dead dye (Life Technologies) and fluorochrome labeled antibodies specific to cell surface markers (BioLegend). After fixation-permeabilization (Fixation/Permeabilization buffer, eBioscience), cells were stained with fluoro-conjugated antibodies specific to IFNγ and granzyme B (BioLegend) or with the isotype control antibodies. Flow cytometry analysis was performed on BD LSRFortessa and data was analyzed by FlowJo V10.
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