Topcount nxt reader

To determine the infectivity of HIV-1 pseudoviruses, TZM-bl cells were seeded into black clear-bottom 96-well plates. The cells were infected with serially diluted pseudoviruses and centrifuged at 4 °C for 30 min at 1550× g to aid virus attachment to cells. The infectivity was measured 48 h post-infection by lysing the samples with the Bright-Glo luciferase substrate (Promega, Fitchburg, WI, USA) at room temperature and immediately reading the luciferase signal using a TopCount NXT reader (PerkinElmer Life Sciences, Shelton, CT, USA). Specific infectivity was obtained by normalizing to the p24 content and plotted as percentage of specific infectivity of viruses produced by cells transfected with an empty vector. The infectivity results were analyzed using an unpaired Student’s t-test implemented in GraphPad Prism version 9.3.1 for Windows (GraphPad Software, La Jolla, CA, USA).

The radiolabeled precursor ³H-thymidine is incorporated into DNA strands during cell replication and radioisotope incorporation is often used as a measure of cell proliferation. All cells were pulsed with 20 μl of RPMI 1640 media containing ³H-thymidine (0.5 μCi) per well and incubated in 5% CO2 at 37 °C for a minimum of 8 h s before harvesting. The cells were then harvested by a Filtermat Harvester (Perkin Elmer) onto Glass fiber matt filters (part No. 6005422) that captures DNA. The filters were then air-dried and placed in Omni Filter cassette and 30 μl of MicroScintTM (Perkin Elmer Cat No 6013611) liquid added, then sealed with TopSeal (Perkin Elmer, Part No 6050195) and loaded on TopCount NXT reader (Perkin Elmer) for determination of incorporation of ³H-thymidine.