To demonstrate that the cocktail of ART used in this study was fully suppressive, PBMC-derived CD4+ T cells from HIV-seronegative donors were activated for 2 days with RP10 containing 10 μg PHA (PeproTech) and 100 IU/mL IL2 (Thermofisher), washed with RP10, and then cultured in 20 IU/mL IL2 for one additional day. The cells were then either left untreated, or pretreated for 1 hr with an ART cocktail consisting of 5 μM AZT, 5 μM Ritonavir, 8 nM Efavirenz, 10 μM Lamivudine, 50 nM Raltegravir, and 0.5 μg/mL T-20 (all from NIH AIDS reagent program). Cells were then cultured for 4 days at a concentration of 2 × 106 cells/mL with RP10 alone or in the presence of 250 ng/mL p24Gag F4.GFP in RP10 containing 20 IU/mL IL2. The cells were then stained for 30 min at 4°C at a concentration of 106 cells/well in FACS buffer, with anti-CD3 APC/Cyanine7, anti-CD4 PE/Cy7, anti-CD8 Alexa Fluor 700, and Zombie Aqua as a Live/Dead discriminator (all from Biolegend). The cells were then washed three times with FACS buffer and fixed overnight in 2% PFA (Electron Microscopy Sciences). The cells were analyzed on an LSRII (BD Biosciences) and Flowjo software was used to gate on live, singlet CD3+CD8- cells and infection rates in these cells were monitored by GFP expression.
Anti cd3 apc cyanine7
Manufactured by BioLegend
Anti-CD3 APC/Cyanine7 is a fluorochrome-conjugated antibody that binds to the CD3 protein expressed on T cells. It can be used in flow cytometry applications to identify and enumerate T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8 alexa fluor 700, by BioLegend (2 mentions)
Live dead discriminator, by BioLegend (2 mentions)
Zombie aqua, by BioLegend (2 mentions)
Anti cd4 pe cy7, by BioLegend (2 mentions)
Phytohemagglutinin (pha), by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Anti f4 80 alexafluor647, by BioLegend (1 mentions)
Anti cd86 pe cyanine7, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti cd4 fitc, by BioLegend (1 mentions)
Anti cd8 pe, by BioLegend (1 mentions)
Anti cd206 fitc, by BioLegend (1 mentions)
Anti cd80 pe, by BioLegend (1 mentions)
Anti cd11b pe, by BioLegend (1 mentions)
Anti cd11c fitc, by BioLegend (1 mentions)
Anti gr 1 apc, by BioLegend (1 mentions)
3 protocols using anti cd3 apc cyanine7
1
ART Suppression of HIV Infection
2
Comprehensive Immune Cell Profiling in Tumor Tissues
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The FACSCanto II flow cytometer (BD Biosciences) was used to detect immune cells in tumor tissues. Single-cell suspensions of tumors were prepared according to the method described by Weigmann et al. [73 (link)]. To analyze CD8+ T cells (CD8+CD3+) and CD4+ T cells (CD4+CD3+), cells were stained with anti-CD3-APC/Cyanine7, anti-CD4-FITC, and anti-CD8-PE antibodies (Biolegend) following the manufacturer’s instructions. For macrophage polarization, cells were stained with anti-CD206-FITC, anti-CD11b-PE, anti-CD86-PE/Cyanine7, and anti-F4/80->Alexafluor 647 antibodies (Biolegend). CD11b+F4/80+CD86+ and CD11b+F4/80+CD206+ cells were defined as M1 and M2 macrophages, respectively. To analyze DC cells (CD11c+CD80+CD86+), cells were stained with anti-CD11c-FITC, anti-CD80-PE, and anti-CD86-PE/Cyanine7 antibodies (Biolegend). To analyze MDSCs (CD11b+Gr-1+), cells were stained with anti-CD11b-PE/Cyanine7 and anti-Gr-1-APC antibodies (Biolegend). Flow cytometry data analysis was conducted using FlowJo 10.8.1 software.
3
Validating ART Cocktail Efficacy
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To demonstrate that the cocktail of ART used in this study was fully suppressive, PBMCderived CD4+ T cells from HIV-seronegative donors were activated for 2 days with RP10 containing 10 μg PHA (PeproTech) and 100 IU/ml IL2 (Thermofisher), washed with RP10, and then cultured in 20 IU/ml IL2 for one additional day. The cells were then either left untreated, or pretreated for 1 h with an ART cocktail consisting of 5 μM AZT, 5 μM Ritonavir, 8 nM Efavirenz, 10 μM Lamivudine, 50 nM Raltegravir, and 0.5 µg/ml T-20 (all from NIH AIDS reagent program).
Cells were then cultured for 4 days at a concentration of 2x10 6 cells/ml with RP10 alone or in the presence of 250 ng/ml p24 Gag F4.GFP in RP10 containing 20 IU/ml IL2. The cells were then stained for 30 minutes at 4°C at a concentration of 10 6 cells/well in FACS buffer, with anti-CD3 APC/Cyanine7, anti-CD4 PE/Cy7, anti-CD8 Alexa Fluor 700, and Zombie Aqua as a Live/Dead discriminator (all from Biolegend). The cells were then washed 3 times with FACS buffer and fixed overnight in 2% PFA (Electron Microscopy Sciences). The cells were analyzed on an LSRII (BD Biosciences) and Flowjo software was used to gate on live, singlet CD3+CD8-cells, and infection rates in these cells were monitored by GFP expression.
Cells were then cultured for 4 days at a concentration of 2x10 6 cells/ml with RP10 alone or in the presence of 250 ng/ml p24 Gag F4.GFP in RP10 containing 20 IU/ml IL2. The cells were then stained for 30 minutes at 4°C at a concentration of 10 6 cells/well in FACS buffer, with anti-CD3 APC/Cyanine7, anti-CD4 PE/Cy7, anti-CD8 Alexa Fluor 700, and Zombie Aqua as a Live/Dead discriminator (all from Biolegend). The cells were then washed 3 times with FACS buffer and fixed overnight in 2% PFA (Electron Microscopy Sciences). The cells were analyzed on an LSRII (BD Biosciences) and Flowjo software was used to gate on live, singlet CD3+CD8-cells, and infection rates in these cells were monitored by GFP expression.
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