HIV Activation Monitoring in Microglial Cells

Flow cytometry analysis was performed to detect the expression of GFP as a signal for HIV activation. HIV infection in HC69.5 cells was assessed by the expression of GFP + cells. To activate the infection, we added 100 µg/ml of Poly IC following previous work published by Alvarez-Carbonell et al.^{12 (link),29 (link)}. Cells were also treated with Δ(9)-THC or CBD at two concentrations (0.01 µM and 1 µM). Cells were collected after 24 h of treatment, fixed, and permeabilized using the Fixation and Permeabilization Solution Kit (BD; Cat. No. 554715). Immortalized human microglial cells (C20, donated by Dr. Karn’s Lab)^{12 (link),29 (link)} were used as uninfected and untreated control. Cells were acquired on an Accuri C6 flow cytometer (BD Accuri; Ann Arbor, MI; Dr. Alejandro Barbieri’s lab, FIU) and analyzed with FlowJo software (Tree Star, Inc.; Ashland, OR). Gating strategy: Forward and side scatter density plots were used to identify interested cell population and to exclude debris. Doublets were excluded by Forward scatter height vs. forward scatter area density plot. Single-parameter histograms were used to identify GFP-positive cells transferring the gate from unactivated cells. Results were expressed as numbers of positive cells.