nonradioactive colorimetric cellular proliferation assay  (Promega)

Journal:

Article Title: Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1 ▿

doi: 10.1128/JVI.01469-06

Figure Lengend Snippet: Effects of cytoskeletal inhibitors on HIV-1 release and viral infectivity. JurkatLAI cells were either left untreated or treated with 1 μM of nocodazole, colchicine, cytochalasin D, or latrunculin for 3 h at 37°C. (A) Cell and viral lysates from untreated or inhibitor-treated cells were separated by SDS-PAGE, and HIV-1 Gag was detected by Western blotting. C, lysates prepared from cell pellets; V, lysates prepared from cell-free virus-containing supernatants. For each condition, 20 μg of cell lysate was used and an equal volume of viral lysate was loaded to allow a direct comparison of virus outputs. The Gag polyprotein p55 and the capsid protein p24 are indicated. (B) Densitometer analysis was performed on Gag p55 and p24 bands from nonsaturated Western blots, and for each treatment condition, the ratio of total cell-free to total cell-associated Gag was calculated. Values are the averages for three independent virus release assays, and the error bars show the SEM. (C) Env incorporation into HIV-1 virions from cells treated with cytoskeletal inhibitors. Supernatants were harvested from JurkatLAI cells that were either left untreated or treated with cytoskeletal inhibitors for 3 h, and virions were concentrated by centrifugation. Viral lysates were separated by SDS-PAGE, and HIV-1 Env was detected by Western blotting with rabbit anti-gp120 serum. A representative blot is shown. (D) Densitometer analysis was performed on gp120 bands from nonsaturated blots. Values are the average pixel intensities of individual bands corresponding to gp120 and are the averages for two independent virus release experiments. Error bars show the SEM. (E) Infectivity assay on viral supernatants harvested from inhibitor-treated cells. JurkatLAI cells were either left untreated (white bar) or treated for 3 h with cytoskeletal inhibitors (gray bars). The cells were then washed to remove inhibitors and incubated for 1 h at 37°C, and virus-containing supernatants were collected and used to infect HeLa reporter cells. Viral infectivity was measured by performing a β-galactosidase assay, and values show the percentages of infectivity relative to that of the untreated control, which was normalized to 100%. Data are the averages for two independent virus release assays, and error bars show the SEM. (F) After 3 h of incubation in the presence or absence of inhibitors, the number of viable JurkatLAI cells was calculated by trypan blue exclusion. Error bars show the standard deviations (SD) for a single experiment performed in triplicate. (G) The metabolic activity of treated or untreated cells was determined using a nonradioactive colorimetric cellular proliferation assay (Promega) that measures bioreduction of the MTS tetrazolium compound. The absorbance at 490 nm was measured, and values are shown for each inhibitor and the untreated control. Error bars show the SD.

Article Snippet: Error bars show the standard deviations (SD) for a single experiment performed in triplicate. (G) The metabolic activity of treated or untreated cells was determined using a nonradioactive colorimetric cellular proliferation assay (Promega) that measures bioreduction of the MTS tetrazolium compound.

Techniques: Infection, SDS Page, Western Blot, Virus, Comparison, Centrifugation, Incubation, Control, Activity Assay, Proliferation Assay