Clariostar fluorescent plate reader

The snap-frozen brain hemispheres were weighed and diluted 3X (m/v) in RIPA buffer (Cell Signalling) with complete protease inhibitor tablet (Sigma-Aldrich), homogenized and lysed by centrifugation at 13000 × g for 90 min at 4 °C. Alexa Fluor 647 fluorescence intensity in the soluble RIPA fraction of brain homogenate was measured using a ClarioStar Fluorescent plate reader (BMG Labtech) and compared to standard curves (Supplementary Fig. 2) of Alexa Fluor 647-conjugated antibody-spiked brain homogenate. The serum concentration was calculated in the same way, except that control mouse serum was used for the standard curves (Supplementary Fig. 3).

Thrombin activity was measured using a Thrombin Activity Assay Kit (Abcam, Cambridge, United Kingdom). Briefly, PRP was incubated with VSV-G or Spike pseudoparticles (1:10) for 10 min at 37°C, followed by stimulation with collagen (30 μg/ml) for 20 min (350 rpm, 37°C). PRP was diluted 1:10 with thrombin assay buffer, and 50 μl of thrombin substrate was added to each well. Thrombin activity was assessed by measuring the conversion of thrombin substrate into its fluorogenic state using a CLARIOstar fluorescent plate reader (BMG Labtech, Ortenberg, Germany) with 350/450 nm excitation and emission filter. Analysis was performed using MARS analysis software.

Activated caspase-3 and 7 activity in hind limb homogenate supernates was determined using the ApoONE fluorescent substrate (Promega, Madison, WI, USA). Briefly, individual, thawed muscle biopsies (80–100 mg biopsy) were mixed with 1.0 mL of 4 °C lysis buffer containing 50 mmol/L Na HEPES, pH 7.4, 100 mmol/L NaCl, 1 mmol/L ethylene diamine tetra-acetic acid, and 10% glycerol and homogenized at 4 °C for 30 s with an Omni tissue homogenizer. The muscle biopsy homogenates were centrifuged at 14,000× g (4 °C) in an Eppendorf microfuge for 20 min. The post-mitochondrial supernates were assayed for cytochrome C and caspase-3,7 activity, and the protein content was determined by the Bradford method using bovine serum albumin (BSA) as the standard. The activated caspase-3,7 assay was performed as follows: 50 μL of muscle supernate (≈150 μg of supernate protein) was mixed with 50 μL of the Apo-ONE substrate solution in the wells of a black, opaque, 96-well plate to initiate the 60 min reaction. The plates were shaken at 200 RPM at 37 °C for 60 min. The DEVD (Asp-Glu-Val-Asp peptide) caspase substrate peptide cleavage was measured using a Clariostar fluorescent plate reader (BMG Labtech, Cary, NC, USA) at an excitation wavelength of 499 nm and an emission wavelength of 521 nm. A caspase internal standard was included in each experiment.