Fixation and permeabilization solution kit

Human pancreatic islets of non-diabetic subjects were cultured with standard medium, or in the presence/absence of IGFBP3 and/or in the presence/absence of Ecto-TMEM219 for 72 h as previously described (see “Pancreatic islets and Interventional studies”). Islets were detached with Versene (ThermoFisher Scientific) and stained with the BD Horizon™ Fixable Viability Stain 510 (FVS510, BD Biosciences 564406, San Jose, CA), which ensures accurate assessment of cell viability in samples after fixation and/or permeabilization as per manufacturer’s instructions. After being stained with BD Horizon™ Fixable Viability Stain 510, cells were next fixed and permeabilized with Fixation and Permeabilization Solution Kit (554714, BD Biosciences, San Jose, CA)^{51 (link)} and finally stained with guinea pig anti-insulin antibody (1:200, Guinea Pig, ThermoFisher Scientific, PA1-26938) followed by AlexaFluor488 anti-guinea pig (1:200, ThermoFisher Scientific, A-11073). Flow cytometry analysis was performed using a BD FACS Celesta flow cytometry system (BD Biosciences) and analyzed using Flowjo software (Version 6 and Version 10, Tree Star, Ashland, OR).

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.

Human pancreatic islets of non-diabetic subjects were cultured with standard medium, or in the presence of IGFBP3 for 72 h as previously described (see “Pancreatic islets and Interventional studies”), and dissociated by trypsinization. In order to select insulin-positive and -negative populations, the cell fraction obtained was permeabilized using the Fixation and Permeabilization Solution Kit (554714, BD Biosciences) and stained with an APC-conjugated anti-insulin antibody (1:100, IC1417A, R&D Systems). Stained cells were flow-sorted using MoFlo Legacy (Beckman Coulter, Brea, CA) and analyzed by qRT-PCR.

Intracellular TNF-α expression was analyzed using a Fixation and Permeabilization Solution Kit with BD GolgiPlug^TM (BD Bioscience). Briefly, THP1 monocytes (1 × 10⁵ cells/ml) or macrophage-like cells (about 2.0 × 10⁵ cells/ml) were exposed to X-rays. At 24 h after X-irradiation, LPS (1 μg/ml) or FSL-1 (50 ng/ml) was added to cultures, and the cells were cultured for an additional 8 h. GolgiPlug, which inhibits protein transporters, was added to the culture medium after 2 h of an 8-h culture. Cells were harvested and fixed using Cytofix/Cytoperm^TM solution (BD Bioscience) for 15 min on ice. After washing twice in Perm/Wash^TM solution (BD Bioscience), cells were suspended in Perm/Wash^TM solution and were stained with anti-human TNF-α-FITC mAbs or isotype control at room temperature in the dark. After 30 min, the cells were washed with Perm/Wash^TM solution and were analyzed using a flow cytometer.