The cytokine secretion ability of B, T, and NK cells was detected using ow cytometry. Brie y, we diluted 100 µL of PBMCs with 400 µL of IMDM (Gibco-BRL, Grand Island, NY, USA) in polystyrene tubes, and then stimulated the cells with or without the leukocyte activation cocktail (BD GolgiPlug™, Germany) for 4 h at 37 ℃ in 5% CO 2 . After stimulation, uorochromeconjugated lineage antibodies (Supplementary Table 1) were added and incubated for 15 min at room temperature (RT) followed by lysis of the RBCs. The cells were then xed and permeabilized with 250 µL of Cyto x/Cytoperm (BD, Germany) at RT for 20 mins. After staining with uorochrome-conjugated cytokine antibodies (Supplementary Table 1) for 20 min at RT, the cell pellets were washed and resuspended in 200 µL PBS. The FACS Canto II ow cytometer (BD Biosciences) and the FACS DIVA software (BD Biosciences) were used for data acquisition and analysis, respectively. The gated lymphocyte populations were further assessed for intracellular cytokine-producing cells. The levels of intracellular cytokines after stimulation were quantitatively determined from the percentage of lymphocytes with higher uorescence intensity than cells without treatment.
Cyto x cytoperm
Manufactured by BD
Sourced in Germany
Cyto x/Cytoperm is a laboratory equipment product designed for cell permeabilization and intracellular staining. It provides a solution for preparing cell samples for flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (3 mentions)
Facsdiva software, by BD (3 mentions)
Facs canto 2 ow cytometer, by BD (3 mentions)
Anti cd45 percp antibody, by BioLegend (2 mentions)
Anti cd3 fitc antibody, by BioLegend (2 mentions)
Anti cd137 apc antibody, by BioLegend (2 mentions)
Anti cd137 mab, by BPS Biosciences (2 mentions)
Prism version 6, by GraphPad (1 mentions)
Verse cytometer, by BD (1 mentions)
Anti hla dr apc clone g46 6, by BD (1 mentions)
Facsverse software, by BD (1 mentions)
Percp cy5.5 anti cd4 clone okt4, by BioLegend (1 mentions)
7 protocols using cyto x cytoperm
1
Cytokine Profiling of Immune Cells
2
Cytokine Profiling of Immune Cells
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The cytokine secretion ability of B, T, and NK cells was detected using ow cytometry. Brie y, we diluted 100 µL of PBMCs with 400 µL of IMDM (Gibco-BRL, Grand Island, NY, USA) in polystyrene tubes, and then stimulated the cells with or without the leukocyte activation cocktail (BD GolgiPlug™, Germany) for 4 h at 37 ℃ in 5% CO 2 . After stimulation, uorochromeconjugated lineage antibodies (Supplementary Table 1) were added and incubated for 15 min at room temperature (RT) followed by lysis of the RBCs. The cells were then xed and permeabilized with 250 µL of Cyto x/Cytoperm (BD, Germany) at RT for 20 mins. After staining with uorochrome-conjugated cytokine antibodies (Supplementary Table 1) for 20 min at RT, the cell pellets were washed and resuspended in 200 µL PBS. The FACS Canto II ow cytometer (BD Biosciences) and the FACS DIVA software (BD Biosciences) were used for data acquisition and analysis, respectively. The gated lymphocyte populations were further assessed for intracellular cytokine-producing cells. The levels of intracellular cytokines after stimulation were quantitatively determined from the percentage of lymphocytes with higher uorescence intensity than cells without treatment.
3
Multiparameter Flow Cytometry of Immune Cells
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Six-parameter ow cytometric analysis was performed on whole blood, BM-, LN-and spleen-derived cells according to standard procedures using a panel of mAbs purchased from BD Biosciences (Pharmingen, San Diego, CA) as follows: anti-CD3-APC-Cy7 (clone SP34), anti-CD8-PE (clone RPA-T8), anti-Ki-67-PE-Cy7 (clone B56), anti-CD14-PE (clone M5E2), anti-CD16-FITC (clone 3G8), and anti-HLA-DR-APC (clone G46-6). The anti-CD4-PerCP-Cy5.5 (clone OKT4) was purchased from Biolegend (San Diego, CA, USA). Isotype antibody was used for a negative control of CD4, Ki67, HLA-DR, CD14 and CD16 expression. Intracellular staining for Ki-67 was performed at room temperature for 30 minutes following permeabilization with cyto x/cytoperm (BD Bioscience). Flow cytometric acquisition and analysis were performed on a BD Verse cytometer driven by the FACS Verse software (BD Biosciences). Analysis of the acquired data was performed using FlowJo software (TreeStar, Ashland, OR, USA) and graphs were prepared using Prism version 6.0 (GraphPad).
4
Cytokine Profiling of Immune Cells
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The cytokine secretion ability of B, T, and NK cells was detected using ow cytometry. Brie y, we diluted 100 µL of PBMCs with 400 µL of IMDM (Gibco-BRL, Grand Island, NY, USA) in polystyrene tubes, and then stimulated the cells with or without the leukocyte activation cocktail (BD GolgiPlug™, Germany) for 4 h at 37 ℃ in 5% CO 2 . After stimulation, uorochromeconjugated lineage antibodies (Supplementary Table 1) were added and incubated for 15 min at room temperature (RT) followed by lysis of the RBCs. The cells were then xed and permeabilized with 250 µL of Cyto x/Cytoperm (BD, Germany) at RT for 20 mins. After staining with uorochrome-conjugated cytokine antibodies (Supplementary Table 1) for 20 min at RT, the cell pellets were washed and resuspended in 200 µL PBS. The FACS Canto II ow cytometer (BD Biosciences) and the FACS DIVA software (BD Biosciences) were used for data acquisition and analysis, respectively. The gated lymphocyte populations were further assessed for intracellular cytokine-producing cells. The levels of intracellular cytokines after stimulation were quantitatively determined from the percentage of lymphocytes with higher uorescence intensity than cells without treatment.
5
CD137 and NF-κB Profiling in GC
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For CD137 detection, the PBMCs or TILs of GC patients were placed in ow tubes, and 5 µL each of anti-CD45-PerCP antibody, anti-CD3-FITC antibody and anti-CD137-APC antibody was added. The cells were incubated in the dark for 10 min and washed with PBS once. Then, 200 µL of PBS was added for ow cytometry detection.
For examination of CD8 + T cells proliferation, CD8 + T cells of GC patients were placed in ow tubes. After washing with PBS once, 200 µL of PBS was added for ow cytometry detection.
For NF-κb detection, CD8 + T cells from GC patients treated with 10 µg/ml agonistic anti-CD137 mAb for 72h were placed in ow tubes. After washing with PBS once, Fixation/Permeabilization Solution (BD Cyto x/Cytoperm TM ) were added at room temperature for 30 min. After washing with PBS once, A NF-κB p65 rabbit mAb (1:1000, CST) was added. The cells were incubated in the dark for 1 h and washed with PBS once. Adding 5 µl anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (1:500, CST) and incubating the cells in the dark for 30 min. After washing with PBS once, 200 µL of PBS was added for ow cytometry detection.
For examination of CD8 + T cells proliferation, CD8 + T cells of GC patients were placed in ow tubes. After washing with PBS once, 200 µL of PBS was added for ow cytometry detection.
For NF-κb detection, CD8 + T cells from GC patients treated with 10 µg/ml agonistic anti-CD137 mAb for 72h were placed in ow tubes. After washing with PBS once, Fixation/Permeabilization Solution (BD Cyto x/Cytoperm TM ) were added at room temperature for 30 min. After washing with PBS once, A NF-κB p65 rabbit mAb (1:1000, CST) was added. The cells were incubated in the dark for 1 h and washed with PBS once. Adding 5 µl anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (1:500, CST) and incubating the cells in the dark for 30 min. After washing with PBS once, 200 µL of PBS was added for ow cytometry detection.
6
Evaluating CD137 and NF-κB in GC Immunology
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For CD137 detection, the PBMCs or TILs of GC patients were placed in ow tubes, and 5 µL each of an anti-CD45-PerCP antibody (BioLegend, USA), anti-CD3-FITC antibody (BioLegend, USA) and anti-CD137-APC antibody (BioLegend, USA) was added. The cells were incubated in the dark for 10 min and washed with PBS once. PBS (200 µL) was added for ow cytometry detection.
For examination of CD8 + T cells proliferation, CD8 + T cells of GC patients were placed in ow tubes and washed once with PBS. PBS (200 µL) was added for ow cytometry detection.
For NF-κB detection, CD8 + T cells from GC patients were treated with 10 µg/ml anti-CD137 mAb (BPS Bioscience, USA) for 72 h and placed in ow tubes. After washing once with PBS, a Fixation/Permeabilization Solution (BD Cyto x/Cytoperm TM ) was added at room temperature for 30 min. After washing once with PBS, an NF-κB p65 rabbit mAb (1:1000, CST, USA) was added. The cells were incubated in the dark for 1 h and washed once with PBS. Five microliters of anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (1:500, CST, USA) was added, and the cells were incubated in the dark for 30 min. After washing once with PBS, 200 µL of PBS was added for ow cytometry detection.
For examination of CD8 + T cells proliferation, CD8 + T cells of GC patients were placed in ow tubes and washed once with PBS. PBS (200 µL) was added for ow cytometry detection.
For NF-κB detection, CD8 + T cells from GC patients were treated with 10 µg/ml anti-CD137 mAb (BPS Bioscience, USA) for 72 h and placed in ow tubes. After washing once with PBS, a Fixation/Permeabilization Solution (BD Cyto x/Cytoperm TM ) was added at room temperature for 30 min. After washing once with PBS, an NF-κB p65 rabbit mAb (1:1000, CST, USA) was added. The cells were incubated in the dark for 1 h and washed once with PBS. Five microliters of anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (1:500, CST, USA) was added, and the cells were incubated in the dark for 30 min. After washing once with PBS, 200 µL of PBS was added for ow cytometry detection.
7
Evaluating CD137 and NF-κB in GC Immunology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For CD137 detection, the PBMCs or TILs of GC patients were placed in ow tubes, and 5 µL each of an anti-CD45-PerCP antibody (BioLegend, USA), anti-CD3-FITC antibody (BioLegend, USA) and anti-CD137-APC antibody (BioLegend, USA) was added. The cells were incubated in the dark for 10 min and washed with PBS once. PBS (200 µL) was added for ow cytometry detection.
For examination of CD8 + T cells proliferation, CD8 + T cells of GC patients were placed in ow tubes and washed once with PBS. PBS (200 µL) was added for ow cytometry detection.
For NF-κB detection, CD8 + T cells from GC patients were treated with 10 µg/ml anti-CD137 mAb (BPS Bioscience, USA) for 72 h and placed in ow tubes. After washing once with PBS, a Fixation/Permeabilization Solution (BD Cyto x/Cytoperm TM ) was added at room temperature for 30 min. After washing once with PBS, an NF-κB p65 rabbit mAb (1:1000, CST, USA) was added. The cells were incubated in the dark for 1 h and washed once with PBS. Five microliters of anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (1:500, CST, USA) was added, and the cells were incubated in the dark for 30 min. After washing once with PBS, 200 µL of PBS was added for ow cytometry detection.
For examination of CD8 + T cells proliferation, CD8 + T cells of GC patients were placed in ow tubes and washed once with PBS. PBS (200 µL) was added for ow cytometry detection.
For NF-κB detection, CD8 + T cells from GC patients were treated with 10 µg/ml anti-CD137 mAb (BPS Bioscience, USA) for 72 h and placed in ow tubes. After washing once with PBS, a Fixation/Permeabilization Solution (BD Cyto x/Cytoperm TM ) was added at room temperature for 30 min. After washing once with PBS, an NF-κB p65 rabbit mAb (1:1000, CST, USA) was added. The cells were incubated in the dark for 1 h and washed once with PBS. Five microliters of anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (1:500, CST, USA) was added, and the cells were incubated in the dark for 30 min. After washing once with PBS, 200 µL of PBS was added for ow cytometry detection.
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