Manager software 6

Plasma samples were collected from HIV-Tg and WT animals at 24 h post-LPS administration and kept frozen until use. Bio-Plex Pro Mouse group 1 Th1 panel L60-00004C6 kit was obtained from Bio-Rad, Portland, ME, USA. Samples from three mice were used for each group. The assay was performed using the Bio-Plex suspension array system according to the manufacturer’s instructions (Bio-Rad). In brief, the appropriate cytokine standards and samples were added to a 96-well flat plate. The samples were incubated at room temperature for 30 min with antibodies chemically attached to fluorescent-labeled magnetic micro beads. After three washes on Bio-Plex ProII wash station, premixed detection antibodies were added to each well and incubated for 30 min. Following three washes, premixed streptavidin-phycoerythrin was added to each well and incubated for 10 min followed by three more washes. Then beads were re-suspended with 125 µL of assay buffer and the reaction mixture was quantified using the Bio-Plex protein array reader. Data were automatically processed and analyzed by Bio-Plex Manager Software 6.0 using the standard curve produced from recombinant cytokine standard.

Detailed clinical assessments were made at baseline and up to 9 months after randomization. Baseline disease severity was assessed by the modified British Medical Research Council (BMRC) grading system [14 ]. An HIV test was performed on all patients at enrollment. Concentrations of total and differential blood cell counts (including CD4⁺ T-cell counts in HIV-infected patients) and CSF leukocyte counts, lactate levels, glucose levels, and protein levels were measured by standard methods.
A panel of 10 human cytokines was measured in stored CSF specimens by Luminex multiplex bead array technology (Bio-Rad Laboratories, Hercules, CA). The panel consists of interleukin 1β (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α). Assays were read on the Bio-Plex 200 platform, and the Bio-Plex Manager software 6.0 was used for bead acquisition and analysis. Additional details on the method for cytokine measurement is provided in the Supplementary Materials.

A total of 48 cytokines including interleukins, chemokines, colony stimulating factors, growth factors, interferons, growth factors and tumor necrosis factor were measured in this study. Cytokine concentrations were measured in serum and human brain tissue TBS extracts using the Pro Human Cytokine Screening Panel 48-plex assay (Bio-Rad, Hercules, California, USA) and 5-point standard curve consisting of S3, S4, S5, S6, S7 standards and a blank, which covered our sample concentration range. Briefly, samples were diluted (serum 1:4, brain tissue 1:2), in sample diluent, and incubated with detection antibodies coupled to magnetic beads, washed using a Bio-Plex Pro wash station and incubated in streptavidin–phycoerythrin before wells were quantified using a Xponent software package (Luminex, Austin, TX). Provided standards generated a five-parameter standard curve for all 48 cytokines and unknown concentrations were calculated with Bio-Plex Manager software 6.1. The intra-assay %CV for the serum plate was 2.68–4.97 (average: 3.51) and for the brain tissue plate 3.65–6.42 (average: 4.86). The inter-assay %CV for the two plates was 4.48–13.57 (average: 7.92).