Memory CD4+ T cells from the blood of two HIV-seronegative donors were purified as described above, and then resuspended at 2 × 107 cells/mL in PBS containing 0.1% FBS. The cells were then loaded for 8 min with 1.5 μM of the proliferation dye CFSE (ThermoFisher). Labeling was then stopped by the addition of an equal volume of pre-warmed, 100% FBS, and the labeled cells were incubated at 37°C for an additional 10 min. The sample was then washed three times in RP10. Memory CD4+ T cells not exposed to CFSE were treated identically in parallel. The time = 0 specimen was immediately stained as described below. The remaining cells were cultured with or without activation with 16 nM PMA, 1 μM ionomycin, and 100 IU/mL IL2 (all three reagents from Thermofisher). At the indicated time points, cells were stained with APC/Cyanine7 anti-human CD3 (Clone SK7, Biolegend), PE/Cy7 anti-human CD4 (Clone A161A1, Biolegend), Alexa Fluor 700 anti-human CD8 (Clone SK1, Biolegend), and Zombie Red (Biolegend) as a Live/Dead discriminator. Stained cells were fixed and analyzed by FACS on an LSRII (BD Biosciences). Flowjo (BD Biosciences) was used for analysis. Live, singlet CD3+CD4+CD8- cells were assessed for proliferation by monitoring the loss of CFSE signal. Results shown are from one of two donors which gave similar results.
Apc cyanine7 anti human cd3
Manufactured by BioLegend
APC/Cyanine7 anti-human CD3 is a fluorescently labeled antibody that binds to the CD3 surface antigen on human T cells. It can be used for the identification and enumeration of T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Pe cy7 anti human cd4, by BioLegend (2 mentions)
Zombie red, by BioLegend (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 700 anti human cd8, by BioLegend (1 mentions)
Percp cyanine 5.5 anti human cd4, by BioLegend (1 mentions)
Pe cyanine7 anti human cd8a, by BioLegend (1 mentions)
Apc anti human cd19, by BioLegend (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
Fitc annexin 5 apoptosis detection kit with pi, by BioLegend (1 mentions)
3 protocols using apc cyanine7 anti human cd3
1
CFSE-Based Proliferation Assay for Memory CD4+ T Cells
2
Multiparametric Flow Cytometry Analysis
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Cells were collected and washed 2 to 3 times. The cell surface mixed antibody was divided equally into each sample staining with the Zombie aqua fixable viability kit (423101, BioLegend), and continued simultaneously for 30 min at room temperature. After the cell surface staining was finished, the intracellular maker staining was performed. Cell fixation (420801, BioLegend) and staining permeabilization (421002, BioLegend) were required prior to intracellular staining. Flow cytometry determined the expression of distinct surface and intracellular molecules. The following antibodies were used: APC/Cyanine7 anti-human CD3 (300317, BioLegend), PerCP/Cyanine 5.5 anti-human CD4 (317427, BioLegend), PE/cyanine7 anti-human CD8a (300913, BioLegend), Brilliant Violet 421 anti-human IFN-γ (502531, BioLegend), and PE/DazzleTM594 anti-human CD107a (LAMP-1) (328645, BioLegend) and APC anti-human TNF-α (502913, BioLegend), APC anti-human CD19 (302211, BioLegend). The apoptosis of BJAB cells was assayed using the FITC Annexin V Apoptosis Detection Kit with PI (640914, BioLegend). The data were analyzedusing FlowJo 10.8 software (https://www.flowjo.com/ ).
3
Proliferation of Memory CD4+ T Cells
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Memory CD4+ T cells from blood of two HIV-seronegative donors was purified as described above, and then resuspended at 2x10 7 cells/ml in PBS containing 0.1% FBS. The cells were then loaded for 8 minutes with 1.5 µM of the proliferation dye CFSE (ThermoFisher).
Labeling was then stopped by addition of an equal volume of pre-warmed, 100% FBS, and the labelled cells were incubated at 37°C for an additional 10 minutes. The sample was then washed 3 times in RP10. Memory CD4+ T cells not exposed to CFSE were treated identically in parallel. The time=0 specimen was immediately stained as described below. The remaining cells were cultured with or without activation with 16 nM PMA, 1 μM ionomycin, and 100 IU/ml IL2 (all three reagents from Thermofisher). At the indicated timepoints, cells were stained with APC/Cyanine7 anti-human CD3 (Clone SK7, Biolegend), PE/Cy7 anti-human CD4 (Clone A161A1, Biolegend), Alexa Fluor 700 anti-human CD8 (Clone SK1, Biolegend), and Zombie Red (Biolegend) as a Live/Dead discriminator. Stained cells were fixed and analyzed by FACS on an LSRII (BD Biosciences). Flowjo (BD Biosciences) was used for analysis. Live, singlet CD3+CD4+CD8-cells were assessed for proliferation by monitoring the loss of CFSE signal.
Results shown are from one of two donors which gave similar results.
Labeling was then stopped by addition of an equal volume of pre-warmed, 100% FBS, and the labelled cells were incubated at 37°C for an additional 10 minutes. The sample was then washed 3 times in RP10. Memory CD4+ T cells not exposed to CFSE were treated identically in parallel. The time=0 specimen was immediately stained as described below. The remaining cells were cultured with or without activation with 16 nM PMA, 1 μM ionomycin, and 100 IU/ml IL2 (all three reagents from Thermofisher). At the indicated timepoints, cells were stained with APC/Cyanine7 anti-human CD3 (Clone SK7, Biolegend), PE/Cy7 anti-human CD4 (Clone A161A1, Biolegend), Alexa Fluor 700 anti-human CD8 (Clone SK1, Biolegend), and Zombie Red (Biolegend) as a Live/Dead discriminator. Stained cells were fixed and analyzed by FACS on an LSRII (BD Biosciences). Flowjo (BD Biosciences) was used for analysis. Live, singlet CD3+CD4+CD8-cells were assessed for proliferation by monitoring the loss of CFSE signal.
Results shown are from one of two donors which gave similar results.
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