Platelet adhesion analysis by LDH assay

Platelets used for adhesion analysis were freshly collected from the blood of two 11- to 13-week-old female Sprague-Dawley rats. Fresh blood collected in a Becton Dickinson (Franklin Lakes, NJ) plasma tube [75 United States Pharmacopeia (USP) units of sodium heparin (spray-coated)] was then immediately centrifuged at 200g for 10 min to get PRP. The residue was further centrifuged at 2000g for 20 min to obtain platelet-poor plasma (PPP). PRP and PPP were gently remixed, and the final platelet density was adjusted to 2 × 10⁸ ml⁻¹. Pre-equilibrated hydrogel disks in PBS (5 mm in diameter and 1 mm in thickness) were placed in 24-well TCPS plates with one disk per well, immersed with 400 μl of final platelet solution, and incubated at 37°C for 3 hours. After incubation, the disks were rinsed five times with 1 ml of PBS and then transferred into new wells. The number of adhered platelets was determined by the lactate dehydrogenase (LDH) assay. For LDH assay, the rinsed samples were soaked in 24-well TCPS plates with one disk per well, 300 μl of PBS, and 10 μl of 10× lysis buffer and incubated at 37°C, 5% CO₂ for 45 min. Fifty-microliter reaction mixtures were then added into each well and incubated at room temperature for 30 min in the dark. To stop the reaction, 50 μl of STOP solution was added to each well and mixed by gentle pipetting. Last, 200 μl of the mixture was taken out from each well, and the absorbance at 490 and 680 nm was measured by a platelet reader. The LDH activity was determined by subtracting the absorbance value of 680 nm from that of 490 nm. Data were normalized to that of TCPS (96-well, control). Each measurement was performed in triplicate (independent replicates).