For surface staining, cells were stained in PBS/0.1% BSA/5 mM EDTA buffer with a fixable viability dye and a combination of the following antibodies: CD45 (30-F11), CD11c (N418), MHCII (M5/114.15.2), F4/80 (BM8), TCRβ (H57-597), all eBioscience; CD103 (M290), CD11b (M1/70), Siglec-F (E50-2440), all BD Biosciences; Ly6G (1A8), Ly6C (HK1.4), CD64 (X54-5/7.1), CD4 (RM4-5), CD24 (M1/69), CD206 (C068C2), all BioLegend. Fc block (anti-CD16/CD32, eBioscience) was included to minimize non-specific antibody binding. For intracellular cytokine staining of T cells, cells were incubated for 3.5 h in complete IMDM medium containing 0.1 μM PMA, 1 μM ionomycin with 1/1000 GolgiPlug and GolgiStop solutions (BD Biosciences), at 37°C in a humidified incubator with 5% CO2. Following surface staining, cells were fixed and permeabilised with the Cytofix/Cytoperm™ Fixation/Permeabilisation Solution Kit (BD Biosciences) according to the manufacturer’s instructions. Cells were stained for 50 minutes with antibodies to IL-17A (17B7, eBioscience) and IFNγ (XMG1.2, eBioscience. All cells were analysed on a SORP LSRII (BD Biosciences) or sorted on a FACSAriaIII (BD Biosciences) to a purity of >95%. Analysis was performed using FlowJo software (Tree Star, Inc).
Cytofix cytoperm fixation permeabilisation solution kit
Manufactured by BD
The Cytofix/Cytoperm™ Fixation/Permeabilisation Solution Kit is a laboratory product used for the fixation and permeabilisation of cells. It is designed to prepare cells for intracellular staining and flow cytometry analysis.
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Lab products found in correlation
Ifn γ xmg1.2, by Thermo Fisher Scientific (1 mentions)
Fc block anti cd16 cd32, by Thermo Fisher Scientific (1 mentions)
Il17a 17b7, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Sorp lsr 2, by BD (1 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
2 protocols using cytofix cytoperm fixation permeabilisation solution kit
1
Flow Cytometry Immunophenotyping Protocol
2
CD8+ T-cell Cytotoxicity Assay
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A CD8+ T‐cell isolation kit (Miltenyl Biotec) was used to separate splenic CD8+ T cells from the newly isolated spleen of 8‐week‐old male Balb/c mice. CD8+ T cells were stimulated for 3 days in RPMI 1640 medium (containing 50 μM β‐mercaptoethanol, antibiotics and 10% FBS) with anti‐CD3 and anti‐CD28 Abs. CT26‐EV and CT26‐SA14 cells (1 × 104 cells/well) were plated in 96‐well plates and incubated for 1 day. After incubation, the cells were cocultured with activated T cells at 2:1 ratio for 12 h (T cells:tumour cells). Before harvest, cells were cultured for 6 h in the presence of brefeldin A (5 μg/ml, BioLegend). Following coculture with cancer cells, collected T lymphocytes were fixed and permeabilised according to the manufacturer's instructions using the Cytofix/Cytoperm fixation/permeabilisation solution kit (BD Bioscience). Thereafter, the cells were stained for 30 min on ice with a FACS buffer‐diluted PE‐conjugated anti‐IFN‐γ Ab (BioLegend) (1:100 ratio). Next, the cells were washed twice with FACS buffer and flow cytometrically examined using a FACSCalibur flow cytometer. The IFN‐γ+CD8+ T‐cell level was calculated using the FlowJo program.
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