Study participants were randomly selected from a prospective community-based cohort to characterize HIV-1 infection in Rakai, Uganda, from 1998 until 2004 [5] (link) prior to the availability of antiretroviral therapy in this setting. Cryopreserved peripheral blood mononuclear cells (PBMCs) from 42 treatment-naive individuals infected with HIV-1 subtype D and 28 community-matched HIV-uninfected controls were selected for study (Table 1 ). Walter Reed Army Institute of Research, Human Subjects Protection Branch (WRAIR#1428), as well as the Uganda National Council of Science and Technology (HS413) approved this study, and all participants provided written consent for participation and for use of stored samples. Plasma viral load was measured using Amplicor HIV-1 Monitor test version 1.5 (Roche Diagnostics, Indianapolis, Indiana, USA). HIV-1 subtypes were determined using the previously described multiregion hybridization assay (MHAacd,[27] (link)). The FACS MultiTEST IMK Kit (BD Biosciences, San Jose, California, USA) was used to enumerate lymphocyte subsets on a dual-laser FACSCalibur (BD Biosciences).
Dual laser facscalibur
Manufactured by BD
Sourced in United States
The Dual-laser FACSCalibur is a flow cytometry system manufactured by BD. It is equipped with two lasers, which allow for the detection and analysis of multiple fluorescent parameters simultaneously. The instrument is designed to perform high-performance cell analysis and sorting tasks.
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2 protocols using dual laser facscalibur
1
Characterizing HIV-1 Infection in Uganda
2
Flow Cytometry Analysis of Lymphocyte Markers
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Flow cytometry analysis was performed as described [22 (link)]. The following antibodies were used to stain cells at 4° C for 20 min: anti-ROR1 mAb (4A5) conjugated with Alexa-647 (4A5-Alexa-647) was generated in our laboratory. Anti-CD23 mAb conjugated with FITC (anti-CD23-FITC) was ordered from Life Technologies (Cat #MHCD2301). Anti-CD200 mAb conjugated with PE (anti-CD200-PE) was obtained from BD Biosciences (Cat #552475). PerCP-Cy5.5 conjugated anti-CD19 was purchased from Ebioscience (Cat #45-0199-42) and PE conjugated anti-CD5 antibodies were from BD Biosciences (Cat, #555355). The stained cells were washed twice with FACS buffer (phosphate buffered saline, pH 7.4 (PBS), 3% FBS), and examined by four-color, multiparameter flow cytometry using a dual-laser FACSCalibur (BD Biosciences), and the data were analyzed using FlowJo software (TreeStar). We subtracted the mean-fluorescence intensity (MFI) of cells stained with a fluorochrome-labeled, isotype-control mAb from the MFI of the same cells stained with each antibody to determine the specific increase in MFI (ΔMFI).
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