Jurkat cells were transduced with VSV-G pseudotyped HIV-1 CH293.1 expressing heterologous Vprs. After 48 hr, cells were fixed with 1% PFA for 20 min at RT and subsequently permeablized with 0.1% Triton to stain p24 (KC57-FITC; Beckman Coulter) for 45 min at 4°C. DNA staining was performed with propidium iodide (50 mg/ml) and 100 μg/ml RNase in 0.1% Triton for 1 hr at RT before flow cytometric analysis to determine the effect of Vpr on cell cycle phases in infected (p24+) cells.
Kc57 fitc
Manufactured by Beckman Coulter
Sourced in United States
The KC57-FITC is a fluorescent-labeled antibody reagent used for the identification and enumeration of CD57-positive cells in human blood samples. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6, by BD (2 mentions)
Macs buffer, by Miltenyi Biotec (2 mentions)
Facscanto 2, by BD (2 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Rockland Immunochemicals (1 mentions)
Rabbit anti flag, by Merck Group (1 mentions)
Rabbit anti est1a smg6, by Abcam (1 mentions)
Mouse anti actin, by Abcam (1 mentions)
Mouse anti ha, by Roche (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Cd8 microbead, by Miltenyi Biotec (1 mentions)
Round bottom ultra low attachment 96 well plate, by Corning (1 mentions)
Kc57 fitc antibody, by Beckman Coulter (1 mentions)
Perm wash buffer, by BD (1 mentions)
Bovine serum albumin (bsa), by Miltenyi Biotec (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Paraformaldehyde (pfa), by BD (1 mentions)
15 protocols using kc57 fitc
1
Vpr-Mediated Cell Cycle Analysis
2
Quantifying HIV-infected MT-4 Cells
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To analyze the amount of productively infected MT-4 cells, cells were washed and fixed in 4% PFA for 90 min, washed twice with PBS, and spun down. Pellets were incubated with HIV Gag p24 flow cytometry antibody KC57-FITC (PN 6604665, Beckman Coulter, Fullerton, CA, USA) at 1:50 dilution in PBS supplemented with 0.1% Triton X-100 and 0.1 mg/ml BSA for 30 min at RT. Subsequently, cells were washed twice, and analyzed using an FACS Verse flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Unstained cells were used as a control for gating. Results were analyzed with the software FlowJo v10 (FlowJo, LLC, Ashland, OR, USA).
3
Antibody Sourcing for p24, UPF, EST1A
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Mouse anti-p24 was obtained from NIH AIDS Reagents Program; rabbit antisera to UPF1 and UPF2 were generously supplied by Jens Lykke-Andersen (University of California, San Diego, CA, USA); rabbit anti-EST1A (SMG6) and mouse anti-actin were purchased from Abcam; rabbit anti-FLAG was purchased from Sigma-Aldrich; mouse anti-HA was purchased from Roche; mouse anti-GAPDH was purchased from Techni-science; mouse anti-nucleolin was purchased from Santa-Cruz Biochemistry; KC57-FITC was purchased from Beckman Coulter; horseradish peroxidase-conjugated secondary antibodies were purchased from Rockland Immunochemicals.
4
HIV Infection Monitoring in Jurkat T Cells
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Jurkat T cells were plated at a density of 2 × 105 cells per well. T20 (50 µM), the V-ATPase inhibitor bafilomycin A1 (100 nM, Sigma Aldrich, St. Louis, USA) or PBS were added 1 h prior to virus challenge. Cells were challenged with VSV-G HIV-1NL4-3ΔEnv and 4 h later, 200 µl of fresh culture medium were added. 48 h later, cells were fixed for 90 min in 4% PFA/PBS and HIV infection was monitored using an intracellular p24 staining (anti-p24 antibody, clone KC57-FITC, Beckmann Coulter, Brea, USA) in principle as reported32 (link).
5
Production and Titration of VSV-G Pseudotyped HIV-1
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Stocks of VSV-G pseudotyped NL4-3 HIV-1 virus were produced by transfecting 4 × 106 HEK293T cells with 3 µg of pNL4-3 and 1 µg of pMD.G (VSV-G) expression vectors using polyethylenimine (PEI) (Polysciences). Virus containing supernatants were harvested 48 h later, filtered and purified by ultracentrifugation on a sucrose cushion. Prior to infection, viral stocks were treated with 100 U/ml of DNase I (Roche Applied Science) for 1 h at 37 °C in the presence of 10 mM MgCl2. Infectious viral titers were assessed by infection of HeLa cells or activated CD4+ T cells with serial dilutions. Twenty-four hpi, percentage of infected cells was determined by FACS analysis by following intracellular capsid (anti-CAp24 antibody, KC57-FITC, Beckman Coulter).
6
HIV Infection Kinetics in CD4+ T Cells
7
Intracellular p24 and Cell Surface Staining
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For intracellular p24gag staining, cells were harvested and fixed in 4% paraformaldehyde for 10 min and diluted to 1% in PBS. Cells were permeabilized in 0.5% Triton-X for 10 min and stained with 1:300 KC57-FITC (Beckman Coulter 6604665; RRID: AB_1575987 ). Data were collected on Accuri C6 (BD Biosciences–U Mass) or a BD FACSCANTO II (Fred Hutch Flow Cytometry Core) and analyzed with FlowJo software. For cell surface marker staining, cells were washed twice in PBS, stained in PBS/1% BSA, incubated at 4°C for 1 hr, washed twice in PBS, and analyzed on the Canto two flow cytometer (Fred Hutch Flow Cytometry Core).
8
Intracellular p24 and Cell Surface Staining
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For intracellular Gagp24 (p24) staining, cells were harvested and fixed in 4% paraformaldehyde for 10 min and diluted to 1% in PBS. Cells were permeabilized in 0.5% Triton-X for 10 min and stained with 1:300 KC57-FITC (Beckman Coulter 6604665; RRID: AB_1575987 ). Cells were read on a BD FACSCANTO II (Fred Hutch Flow Cytometry Core) and analyzed in FlowJo. For cell surface marker staining, cells were washed twice in PBS, stained in PBS/1% BSA, incubated at 4°C for 1 hr, washed twice in PBS, and analyzed on the Canto two flow cytometer (Fred Hutch Flow Cytometry Core).
9
Cell Surface Expression of HIV Env
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For cell surface staining, HeLa cells were plated at a density of 400,000 per well of a six well plate and transfected with 400 ng of mutant Env. 24 hours post transfection, cells were stained with zombie violet dye (BioLegend/Perkin Elmer) diluted 1:500 in PBS, then fixed with 4% paraformaldehyde. Cells were washed twice with PBS then blocked with Dako blocking reagent, followed by staining on the cell surface with 2G12 anti-gp120 directly conjugated to APC (Abcam, Cambridge, UK; ab201807) diluted 1:100 in Dako antibody diluent for 2 hours. 2G12 was washed out with PBS twice followed by permeabilization with 0.2% Triton X-100 and staining for intracellular Gag with KC57-FITC (Beckman Coulter, Pasadena, CA; 6604665) diluted 1:100 in Dako antibody diluent. Cells were washed with PBS and resuspended in MACS buffer (Miltenyi Biotec, North Rhine-Westphalia, Germany) followed by analysis using BD FACS Canto II. Alternatively, 5 million H9 cells were infected overnight with appropriate viral mutant with 1ug of p24 in 1 mL to ensure a high degree of infection. H9 cells were washed once with PBS the next day and incubated with 2 mL RPMI for an additional 24 hours. The next day, cells were processed as described previously.
10
HIV-1 Infection Time Course Assay
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Mixed cell suspensions were infected with HIV-1 BaL for 2h at a MOI of 0.1 after which residual virus was washed away. The same input number of cells was used for EM, END and ECT. Uninfected controls were incubated with medium without the virus for the same amount of time. After incubation, cells were plated in round bottom ultra low attachment 96-well plates (Corning, Corning, NY). Cell cultures were maintained for 6–7 days, with half of the well media collected and replaced with fresh media on day 3. At the end of the infection time, cells were washed, stained for surface markers as indicated and levels of p24 measured intracellularly by flow cytometry KC57-FITC; Beckman Coulter). Additionally, released p24 into the culture media was measured by p24 ELISA (Advanced Bioscience Laboratories, Rockville, MD) following manufacturer’s recommendations.
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