Viral fusion was assessed as described [54 (link),55 (link)]. Briefly, ultracentrifuged HIV-2 virions containing the Blam-Vpr2 fusion protein (a gift of Matthias Dittmar, [55 (link)]) were used to infect 1.5 × 105 MDDCs in 100 μL at 37°C, in presence of 10 mM Hepes and 2 mg.mL-1 DEAE Dextran (Sigma). After 2 hours, cells were washed in cold CO2-independent medium (Invitrogen), without FBS, resuspended in cold CO2-independent medium supplemented with 10% FBS and incubated with the CCF2-AM substrate (CCF2-AM kit, Invitrogen), in the presence of 1.8 mM Probenecid (Sigma), for 2 hours at room temperature. Cells were extensively washed in cold CO2-independent medium and fixed in PBS-PFA 4% for 5 min. The cleaved CCF2-AM fluorescence (excitation at 405 nm, emission at 450 nm) was then immediately measured by flow cytometry using the DAPI channel, on a FacsCanto II (BD).
Ccf2 am kit
Manufactured by Thermo Fisher Scientific
The CCF2-AM kit is a fluorescence-based assay used to measure intracellular calcium levels in live cells. The kit contains the cell-permeable calcium indicator dye CCF2-AM, which can be loaded into cells and then detected using a fluorescence microscope or plate reader.
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2 protocols using ccf2 am kit
1
Viral Fusion Assay in MDDCs
2
Measuring Viral Fusion Efficiency
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Viral fusion was assessed as described (Casartelli et al., 2010 (link), Cavrois et al., 2002 (link)). Briefly, virions containing the βlam-Vpr fusion protein were produced by cotransfecting 293T cells with a 3:1.5:1 ratio of pNL4-3, FLAG-IFITM3, and βlam-Vpr plasmids, respectively. A total of 25 ng Gag p24 of βlam-Vpr-containing virions was exposed to 1 × 105 SupT1 or MT4C5. After 2 hr, cells were incubated with the CCF2-AM substrate (CCF2-AM kit, Invitrogen) for 2 hr. The cleaved CCF2-AM fluorescence was then measured by flow cytometry. The virus-target cell fusion assay was performed as described (Casartelli et al., 2010 (link)).
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