To determine the effect of IN.mScarlet incorporation on virus infectivity, SupT1-R5 cells were distributed into 96-well plates (3 × 105 cells/well; U-bottom; Greiner Bio-One, 650180) and infected with equal amounts of non-labeled or IN.mScarlet carrying wild-type HIV-1NL4-3 virions (both at 1 μUnits of RT/cell). To determine HIV-1 infectivity upon CPSF6 downregulation, SupT1-R5 cells with AAV (expressing CPSF6 shRNA or non-silencing control shRNA) were distributed at 72 h post-transduction into 96-well plates (1 × 105 cells/well; U-bottom; Greiner Bio-One) and infected with wild-type HIV-1 (1 μUnits of RT/cell). Infected cells were incubated for 24 h at 37°C before addition of T-20 (50 μM) fusion inhibitor to prevent a second round of infection. Infectivity was scored at 48 h p.i. by flow cytometry. For this, cells were fixed with 4% FA in PBS for 90 min at room temperature and immunostained for intracellular HIV-1 CA for 30 min at 4°C using KC57-FITC antibody (Beckman Coulter) diluted in 0.1% Triton X-100, 0.1 mg/ml BSA in PBS. Cells were analyzed using a BD FACSCelesta flow cytometer (BD Biosciences).
Kc57 fitc antibody
Manufactured by Beckman Coulter
The KC57-FITC antibody is a fluorescently-labeled monoclonal antibody that recognizes the p24 antigen of the human immunodeficiency virus (HIV). It is used in flow cytometry applications for the detection and quantification of HIV-infected cells.
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Lab products found in correlation
Cd8 fitc, by Thermo Fisher Scientific (2 mentions)
Cd3 apc, by Thermo Fisher Scientific (2 mentions)
Cd4 pe, by Thermo Fisher Scientific (2 mentions)
Cd3 apc cy7, by Thermo Fisher Scientific (2 mentions)
Ccr5 pe, by R&D Systems (2 mentions)
Ccr6 percp cy5, by BioLegend (2 mentions)
Cd90 alexa fluor 647, by BioLegend (2 mentions)
Ccr5 pe cy7, by BD (2 mentions)
Cd4 fitc, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Facscanto, by BD (2 mentions)
Cytofix cytoperm plus kit, by BD (2 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Kc57 fitc, by Beckman Coulter (1 mentions)
Perm wash buffer, by BD (1 mentions)
4 protocols using kc57 fitc antibody
1
Determining HIV-1 Infectivity via Flow Cytometry
2
HIV Infection Analysis by Flow Cytometry
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Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD8-FITC, CD4-FITC, CD4-PE, CD3-APC, CD3-APC-Cy7 (eBioscience, San Diego, CA), CCR5-PE-Cy7 (BD Biosciences, San Jose, CA), CCR5-PE (R&D Systems, Minneapolis, MN), CCR6-PerCp-Cy5.5, CD90 Alexa Fluor-647 (Biolegend, San Diego, CA). For the HIV-infection experiments, following infection for 6–7 days, intracellular levels of p24 were analyzed as described previously8 (link). Briefly, after surface staining, cells were washed, fixed and permeabilized (20 min) following instructions provided in the Cytofix/Cytoperm Plus kit (BD Biosciences) and stained for intracellular p24 with KC57-FITC antibody (Beckman Coulter; Danvers, MA) for 30 min. Analysis was performed on BD FACSCalibur or BD FACSCanto flow cytometers (BD Biosciences) using FACSdiva software, and data analyzed with FlowJo software (Tree Star, Inc. Ashland, OR). Expression of surface markers was measured by the percentage of positive cells and the mean fluorescence intensity (MFI).
3
HIV Infection Analysis by Flow Cytometry
4
Intracellular HIV p24 Antigen Detection
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To analyze cells actively infected with HIV, we stained for intracellular p24 antigen (KC57-FITC; Beckman Coulter) and permeabilized using the BD Fixation/Permeabilization Kit (catalog #554714) according to the manufacturer's instructions. Cells were fixed using 4% paraformaldehyde at 4°C for 20 min and washed twice with staining buffer (Dulbecco's PBS without Mg2+ or Ca2+, 1% heat-inactivated FCS, 0.09% (w/v) sodium azide, pH adjusted to 7.4). For permeabilization, cells were incubated in BD Perm/Wash buffer (BD Biosciences) for 15 min and stained with KC57-FITC antibody (Beckman Coulter). Because we performed intracellular p24 staining in combination with cell surface antigen staining (as in Figures 5 C and 5D), we first stained with the fluorochrome-conjugated monoclonal antibodies against CCR5 and CXCR4 before proceeding to the fixation and permeabilization steps.
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