For detection of intracellular IFNγ and TNFα, lymphocytes were stimulated for 4 hours with the indicated tumor cells (effector-to-target ratio of 1:1 or 1:2) in AIM-V complete medium containing 10 μg/mL Brefeldin A (Sigma-Aldrich). Then cells were stained with antihuman CD3-PE/Cy7 and CD8-APC-Alexafluor700 antibodies (Beckman Coulter) followed by fixation and permeabilization using the Fixation/Permeabilization Concentrate and Diluent Kit (eBioscience) and addition of an anti-IFNγ–FITC antibody (Beckman Coulter) or anti-TNFα–Pacific Blue antibody (Biolegend). Cells were analyzed in a Gallios flow cytometer, and the Kaluza software was used for data analysis (Beckman Coulter). Where indicated, antibody W6/32 (50 μg/mL; purified from hybridoma supernatant, kindly provided by M. Fatho, Mainz) and anti–PD-L1 (10 μg/mL; Biolegend) were added to block the TCR/HLA class I and PD-1/PD-L1 interactions, respectively. Mouse monoclonal IgG1 (mIgG; R&D Systems) was used as control antibody.
Fixation permeabilization concentrate and diluent kit
Manufactured by Thermo Fisher Scientific
The Fixation/Permeabilization Concentrate and Diluent kit is a laboratory reagent used in the preparation and processing of samples for flow cytometry analysis. The kit provides the necessary components to fix and permeabilize cells, allowing for the intracellular staining of cellular proteins and other molecules.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Brefeldin a, by Merck Group (2 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Anti pd l1, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
B220 fitc, by Thermo Fisher Scientific (1 mentions)
Annexin 5 and pi, by BD (1 mentions)
Facsaria, by Tree Star (1 mentions)
Facscallibur, by BD (1 mentions)
Live dead zombie, by BioLegend (1 mentions)
Facsaria iiiu, by BD (1 mentions)
Cd8 apc cy7, by BioLegend (1 mentions)
Anti human cd3 brilliant violet 421, by BioLegend (1 mentions)
7 protocols using fixation permeabilization concentrate and diluent kit
1
Intracellular Cytokine Staining Assay
2
Immunophenotyping of T Cell Subsets
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Single-cell suspensions were prepared. Samples were stained (20–30 min) with the following antibodies: anti-CD45, anti-CD3, anti-CD4, anti-CD8α, anti-CD25 antibodies. For intracellular staining, cells were fixed, permeabilized overnight at 4 °C (Fixation/Permeabilization Concentrate and Diluent kit, eBioscience, San Diego. CA) and subsequently stained using anti-Foxp3 antibody for 30 min. All experiments were performed on BD FACSCalibur or BD LSRFortessa and data was analyzed with FlowJo 7.6.1.
3
Comprehensive Immune Cell Profiling
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Lymphocytes from each organ was stained with various combinations of mAbs to CD4-FITC/APC, CD8α-PE/APC, CD3α-PE, and B220-FITC (eBiosciences), CD103-APC, LAP (TGF-β1)-PE (Clone TW7-20B9, Biolegend). Fixation/Permeabilization Concentrate and Diluent Kit (eBiosciences) were used for the intracellular staining of FoxP3-PE/APC according to the user’s handbook. After stained with surface markers, cells were stained with Annexin V and PI (BD Pharmingen) to examine apoptosis. For in vivo BrdU incorporation assay, mice were administered of BrdU (16 mg/ml; 50 μl) via i.n. route [48 (link)]; cells were harvested after 24hours and stained with BD FastImmune BrdU kit (BD Biosciences). Samples were run on BD Biosciences FACSCalibur, FACSAria or Accuri C6 instruments, and analyzed with FlowJo software (Tree Star, Ashland, OR).
4
Multiparameter flow cytometry analysis
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Single-cell suspensions were prepared as mentioned previously55 (link). Samples were stained (20–30 min) with the following antibodies: anti-CD45, anti-CD3, anti-CD4, anti-CD8a, anti-CD25, anti-CD11b, anti-Gr-1, anti-MHC I, anti-CXCR3, and anti-CCR5 antibodies. For intracellular staining, cells were fixed, permeabilized overnight at 4 °C (Fixation/Permeabilization Concentrate and Diluent kit, eBioscience), and subsequently stained using anti-IFN-γ or anti-Foxp3 and analyzed by flow cytometry (BD FACSCallibur). All antibodies were collected from eBioscience (San Diego, CA, USA) or BioLegend (USA).
5
Comprehensive Immune Cell Phenotyping
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All antibodies were purchased from BD Biosciences, eBioscience, Biolegend and R&D system (listed in Supplementary Table S1 ). For surface staining, cells were stained with fluorescent labelled antibodies in PBS with 2% FCS for 30 minutes on ice. Viability was assessed by staining with fixable Live/Dead Zombie (Biolegend) or DAPI. For intracellular staining, cells were seeded (1 × 106 cells per well) in 96-well U-bottomed plates and stained with antibodies against surface markers, fixed with 2% PFA for 10 minutes at 25 °C, permeabilized with 0.2% Saponin and then stained with anti-FOXP3, anti-IFNγ and anti-granzyme B using Fixation/Permeabilization Concentrate and Diluent kit (eBioscience). Samples were analyzed on a BD Fortessa flow cytometer and FlowJo software (Treestar, version 10.7.2).
6
Tumor Tissue Immune Profiling
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Tumor tissues were collected, minced into small pieces, and digested in 2 mg/ml collagenase Type IV at 37°C for 1 h. The digested tumor tissues were then filtered through a 70 μm cell strainer to make a single-cell suspension. Surface markers of cell samples were stained at 4°C for 30 min with antibodies: anti-CD45, anti-CD3, anti-CD4, anti-CD8α, anti-PD-1, anti-PD-L1. For intracellular staining, cells were fixed, permeabilized overnight at 4°C (Fixation/Permeabilization Concentrate and Diluent kit, eBioscience) and subsequently stained with anti-IFN-γ antibody. All the samples were analyzed on the FACSCalibur or FACSAria IIIu (BD Biosciences) flow cytometer and the data were analyzed with FlowJo 10.0.8 software.
7
Quantifying Tumor-Infiltrating Lymphocyte Activation
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A total of 1 Â 10 5 TILs were coincubated for 4 hours with the indicated autologous tumor cells (1 Â 10 5 cells) in AIM-V complete medium containing 10 mg/ml brefeldin A (Sigma-Aldrich). Cells were then fixed and permeabilized using the Fixation/Permeabilization Concentrate and Diluent kit (eBioscience) followed by staining with an antibody cocktail containing antihuman CD3-Brilliant-Violet-421, CD8-APC-Cy7, TNFa-PE-Cy7, and IFN-g-PE antibodies (BioLegend). Cells were analyzed on a Gallios flow cytometer, and the Kaluza software was used for data analysis, Gating strategy is shown in Supplementary Figure S5b.
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