HIV Viral Fusion Assay in DCs

Viral fusion was analyzed as described [29 (link)]. Briefly, day 5 DCs were plated into a 96-well plate in triplicates (1.5x10⁵ cells/100μl) in RPMI in presence of 10mM Hepes (Life Technologies) and 2mg/ml DEAE-Dextran (Sigma-Aldrich). Cells were exposed to the indicated concentrations of non-opsonized (HIV) or complement-opsonized (HIV-C) HIV-1 containing Blam-Vpr. After 3 hours, cells were washed 2 times in CO₂-independent medium (Life Technologies), re-suspended in CO₂-independent medium containing 10% FCS and loaded with the CCF2-AM substrate solution (LiveBLAzer FRET-B/G Loading Kit with CCF2-AM, Life Technologies). After 2h incubation at room temperature (dark) cells were washed 2 times in CO₂-independent medium, fixed in 4% paraformaldehyde for 30 min and respective wells were pooled for flow cytometric analysis.

Free full text: Click here

Posch W., Steger M., Knackmuss U., Blatzer M., Baldauf H.M., Doppler W., White T.E., Hörtnagl P., Diaz-Griffero F., Lass-Flörl C., Hackl H., Moris A., Keppler O.T, & Wilflingseder D. (2015). Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells. PLoS Pathogens, 11(6), e1005005.

Publication 2015

Hepes DEAE-Dextran CO2-independent medium LiveBLAzer FRET-B/G Loading Kit with CCF2-AM

Cells Deae dextran Flow cytometric Fret Hepes Hiv vpr Paraformaldehyde

Corresponding Organization : Sorbonne Université

Top 5 similar protocols

Variable analysis

independent variables

Concentration of non-opsonized (HIV) or complement-opsonized (HIV-C) HIV-1 containing Blam-Vpr

dependent variables

Viral fusion

control variables

Day 5 DCs plated into a 96-well plate in triplicates (1.5x10^5 cells/100μl) in RPMI in presence of 10mM Hepes (Life Technologies) and 2mg/ml DEAE-Dextran (Sigma-Aldrich)
Cells were exposed to the indicated concentrations of non-opsonized (HIV) or complement-opsonized (HIV-C) HIV-1 containing Blam-Vpr
After 3 hours, cells were washed 2 times in CO2-independent medium (Life Technologies), re-suspended in CO2-independent medium containing 10% FCS and loaded with the CCF2-AM substrate solution (LiveBLAzer FRET-B/G Loading Kit with CCF2-AM, Life Technologies)
After 2h incubation at room temperature (dark) cells were washed 2 times in CO2-independent medium, fixed in 4% paraformaldehyde for 30 min and respective wells were pooled for flow cytometric analysis

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!