Synergy mx fluorescence plate reader

The expression of nitrite (NaNO₂), a stable end product resulting from the reaction of NO with molecular oxygen, was measured in supernatants collected from treated and untreated microglia using a Griess reagent system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. A synergy Mx fluorescence plate reader (Bio-Tek Instruments) was used to detect the absorbance at 550 nm. The concentration of nitrite was calculated from a standard curve using sodium nitrite solution.

Boyden chambers (Corning Costar, NY, United States) were used to determine the transmigration of monocytes in vitro. Briefly, primary HBMECs were seeded (4 × 10⁴ cells/well) onto 6.5-mm polyester transwell inserts (3-μm pore size) and grown for 5days to achieve confluence. Human monocytes were pretreated with/without CXCR3 inhibitor AMG487 or TBK1 inhibitor Amlexanox for 1 h, followed by exposure of cells to HIV Tat protein for an additional 24 h and subsequently fluorescently labeled with 10 μM cell tracker green for 10 min at 37°C. Labeled cells (1 × 10⁶ cells/ml) were added to the upper compartment of transwell inserts in serum-free medium, and the chemokine CXCL10 was added to the basal side of the chamber at a final concentration of CXCL10 at 100 ng/ml. The transwell plates were incubated for 18 h at 37°C, followed by quantification of monocyte transmigration by measuring the number of migrated cells across the insert using a Synergy Mx fluorescence plate reader (BioTek Instruments, Winooski, VT, United States).

FECH activity was measured by aerobic enzymatic formation of zinc-protoporphyrin IX (Zn-PpIX) using a modified van Hillegersberg method, as described previously^{45 (link),46 (link)}. Briefly, cell lysates were incubated with 200 µM PpIX (Sigma Aldrich) in 200 µL assay buffer (0.1 M Tris–HCl, 1 mM palmitic acid (Sigma Aldrich) and 0.3% v/v Tween 20, pH 8.0) and then 50 µL 2 mM zinc acetate solution was added. The mixture was incubated at 37 °C for 60 min. The reaction was terminated by adding 500 µL ice-cold stop buffer (1 mM ethylenediaminetetraacetic acid (EDTA) in 30:70 DMSO/ methanol). The reaction mixture was centrifuged at 14,000 × g for 10 min, and Zn-PpIX in the supernatant was measured using a Synergy Mx Fluorescence plate reader (BioTek Instruments Inc. VT) with a 405 nm excitation/590 nm emission filter. Heat-inactivated cell lysates were included as a negative control.

For evaluating changes in cell cycle progression and PCD-induced DNA fragmentation after PDT, the cells were harvested 4 h after 5-ALA-PDT, fixed and permeabilized with 70% cold ethanol, and then stained with propidium iodide (PI) solution (50 µg/ml PI in PBS with 550 U/ml RNaseA; Abcam, USA). Cellular DNA content was analysed by flow cytometry using a BD FACSCalibur (BD Biosciences, San Jose, CA, USA).³⁵ The data were analysed using FlowJo (FlowJo LLC, OR). Western blot analysis was conducted to detect PCD markers (cleaved PARP, pro-caspase 3 and cleaved caspase 3) using the apoptosis western blot cocktail (ab136812, Abcam).^{36 (link)} The amount of cellular ROS was measured using the OxiSelect^TM Intracellular ROS assay kit (Cell Biolabs Inc. San Diego, CA, USA) following the manufacturer’s instructions. Briefly, 5-ALA-PDT-treated cells were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min at 37 °C, and the fluorescence was measured using a Synergy Mx Fluorescence plate reader (BioTek Instruments Inc., VT) at 480 nm/530 nm. The amount of ROS was determined by comparison with a 2′,7′-dichlorodihydrofluorescein (DCF) standard curve.