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Schindler et al. Microparticles in neuroimmune signaling

DMEM/F12 with 5% FBS. Following 30-min incubation, in the MP samples was calculated. MPs were used in
cells were stimulated by exposing them in duplicate wells experiments at a protein concentration of 10 μg/mL.
to TFAM (2.5 μg/mL), IFN-γ (150 U/mL), IFN-γ plus TFAM
(0.5 μg/mL), IFN-γ plus LPS (0.5 μg/mL), or vehicle Toxicity of MP-stimulated monocytic THP-1
solution (phosphate buffered saline, PBS). Following cells towards neuronal SH-SY5Y cells
a 24-h incubation period, cultured supernatants from To study the cytotoxicity of monocytic THP-1 cells
each well were collected into individual 50 mL centrifuge induced by MPs, supernatant transfer experiments
[35]
tubes and total cell numbers from each treatment were performed as previously described. Briefly,
were counted using a hemocytometer. A differential THP-1 cells were seeded into 96-well plates as 250 μL
centrifugation procedure consisting of three steps was aliquots at a concentration of 0.5 million cells/mL in
then performed at 4°C: (1) 5 min at 300 g; (2) 20 min DMEM/F12 with 5% FBS. Following 30 min incubation,
at 1,200 g; (3) 30 min at 10,000 g. After the first two THP-1 cells were stimulated with IFN-γ (150 U/mL),
centrifugation steps, the supernatants were collected THP-1-derived MPs (10 μg protein/mL), IFN-γ plus
and transferred into new 50 mL tubes. After the third THP-1-derived MPs, or left unstimulated by adding MP
step, however, the supernatants were discarded. The vehicle solution (1X Annexin-V binding buffer). After 48 h
pellets were re-suspended in 1 mL of sterile PBS and incubation, THP-1 cell supernatants were transferred
the MP samples were washed by centrifugation for onto SH-SY5Y cells seeded 24 h earlier into 96-well
30 min at 10,000 g. The MP pellet was re-suspended in plates at 0.2 million cells/mL in 200 μL DMEM/F12
1X Annexin-V binding buffer from the FITC Annexin-V with 5% FBS. In addition, THP-1 cell supernatants
Apoptosis Detection Kit. The MP samples were stored were collected for IL-6, TNF-α, and MCP-1 ELISA
at -20°C. measurements. THP-1 cell viability was assessed
using the MTT assay. SH-SY5Y cells were incubated
MP detection and quantification for additional 72 h at which point an MTT assay was
Flow cytometry performed to assess their viability.
To detect and quantify MPs released by THP-1 cells,
the MP samples were analyzed using flow cytometry Direct toxicity of MPs derived from stimulated
according to previously described methods. Briefly, THP-1 cells towards neuronal SH-SY5Y cells
[32]
0.1 mL from each MP sample was combined with To determine whether the MPs isolated from stimulated
5 μL of Annexin-V-FITC stain and incubated at room human monocytic THP-1 cells were directly toxic
temperature in the dark for 15 min. Annexin-V was to human neuronal SH-SY5Y cells, the following
used as it binds to the externalized phosphatidylserine experiment was performed. Human neuronal SH-SY5Y
on the surface of MPs. Following incubation, cells were seeded into 24-well plates as 400 μL aliquots
[33]
samples were centrifuged for 30 min at 10,000 g to per well at a concentration of 0.2 million cells/mL in
remove any unbound Annexin-V-FITC. The MP pellet DMEM/F12 with 5% FBS. Following 24-h incubation,
was resuspended in 0.1 mL of 1X Annexin-V binding SH-SY5Y cells were treated with MP vehicle solution
buffer and analyzed using the MACSQuant Analyzer (1X Annexin-V binding buffer) or MPs (10 μg protein/mL)
10 with MACSQuantify software (Miltenyl Biotec). The isolated from unstimulated THP-1 cells or THP-1 cells
MP size gate was defined using 0.5 μm and 1.0 μm that had been stimulated with IFN-γ in combination
calibration beads and events within the MP gate were with LPS. Following 72-h incubation of SH-SY5Y cells
further discriminated by Annexin-V label. A logarithmic with the MPs, the MTT assay was performed to assess
scale was used for side scatter, forward scatter, neuronal cell viability.
and fluorescence channels. MPs were identified as
Annexin-V positive events within the MP size gate MTT cell viability assay
with fluorescence intensity above a control sample not Viability of the cells used during experiments was
stained with Annexin-V-FITC. assessed using the MTT assay as previously
described. [36,37] This assay is based on the ability of
BCA protein assay viable cells to reduce the water-soluble tetrazolium
Protein concentration in the MP samples was dye MTT to an insoluble purple formazan product,
measured using the BCA protein assay as previously which can be measured spectrophotometrically.
described. Briefly, 10 μL of each MP sample were MTT (0.5 mg/mL) was added to the wells containing
[34]
analyzed according to the instructions provided for the cultured cells and the plates were incubated for 1 h at
Pierce BCA Protein Assay Kit. BSA standards (0.0125- 37°C. A 20% SDS/50% DMF solution was then added
2 mg/mL) prepared by diluting the BSA stock solution at a 1:1 ratio to each well to solubilize the formazan
(2 mg/mL) in distilled water were used to construct a crystals. After an additional incubation period of 3-4 h at
standard curve from which the protein concentration 37°C, 0.1 mL aliquots from each well were transferred
234 Neuroimmunology and Neuroinflammation ¦ Volume 3 ¦ October 28, 2016