Author: Wilkins, Jordan; Zheng, Yi-Min; Yu, Jingyou; Liang, Chen; Liu, Shan-Lu
Title: Nonhuman Primate IFITM Proteins Are Potent Inhibitors of HIV and SIV Document date: 2016_6_3
ID: 1m1woscb_21
Snippet: The fusion assay was performed as previously described [43] . HEK293T cells were seeded onto 60 mm plates and cotransfected with 3 μg of HIV-1-NL4.3 and 1 μg of pCMV-BlaM-Vpr; virions were harvested 48-72 h post-transfection. SupT1 cells were incubated with virus and spinoculated at 4˚C for 1 hour and then further incubated at 37˚C for 3 hours. SupT1 cells were resuspended in CO2-independent media and loaded with CCF2 dye according to manufac.....
Document: The fusion assay was performed as previously described [43] . HEK293T cells were seeded onto 60 mm plates and cotransfected with 3 μg of HIV-1-NL4.3 and 1 μg of pCMV-BlaM-Vpr; virions were harvested 48-72 h post-transfection. SupT1 cells were incubated with virus and spinoculated at 4˚C for 1 hour and then further incubated at 37˚C for 3 hours. SupT1 cells were resuspended in CO2-independent media and loaded with CCF2 dye according to manufacturer's recommendations (Invitrogen). Cells were washed and incubated in CO2-independent media with 10% FBS and 0.5 μM probenecid for 16 hours at room temperature. Samples were fixed in 2% formaldehyde, washed, and resuspended in PBS. Uncleaved CCF2-AM dye was detected at Ex409/Em518 and cleaved CCF2 was detected at Ex409/Em447 using a BD Fortessa flow cytometer.
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