Dri chem slide ldh piii

This study was performed in accordance with the Declaration of Helsinki, and the Institutional Review Board of Taipei Medical University approved all protocols (IRB: N201612050). All volunteers provided informed consent before they participated in this study. Anticoagulated human blood with acid–citrate–dextrose (1:9) was collected from healthy human volunteers who had not eaten any drugs within a time of two weeks prior to the analysis. The method described by Sheu et al. [20 (link)] was used for preparing human platelet suspensions. The platelets were suspended in Tyrode’s solution, and calcium chloride was then added, with the final concentration of Ca²⁺ being 1 mM.
The cytotoxicity of the CDOTs was evaluated using an LDH release assay. Washed platelets (3.6 × 10⁸ cells/mL) were pretreated with 50-500 μM CDOTs or a solvent control (PBS; phosphate-buffered saline) for 20 min at 37 °C and then centrifuged at 5000 g for 5 min. The supernatant obtained was used for the assay. Briefly, 10 μL of the supernatant was placed on a Fuji Dri-Chem slide (LDH-PIII) (Tokyo, Japan), and the absorbance was measured at 540 nm using a UV–vis spectrophotometer (UV-160; Shimadzu, Japan). The LDH activity of 1% Triton X-100-treated washed platelets indicated 100% LDH release.

To measure the intracellular calcium [Ca²⁺]i, citrated whole blood was centrifuged, and the supernatant was incubated with 5 μM Fura 2-AM, which was then measured using a Hitachi Spectrometer F-7000 (Tokyo, Japan). [Ca²⁺]i was measured at excitation wavelengths of 340 and 380 nm and an emission wavelength of 500 nm [7 (link)]. Furthermore, cytotoxic effect was examined by determining the level of lactate dehydrogenase (LDH). Washed platelets were preincubated with CBN (60, 80, and 160 μM) or 0.1% DMSO for 20 min at 37 °C. An aliquot of the supernatant (10 μL) was deposited on a Fuji Dri-Chem slide LDH-PIII (Fuji, Tokyo, Japan), and the absorbance was read using a spectrophotometer (UV-160; Shimadzu, Japan). The maximal value of LDH was observed in triton-treated platelets.

Washed platelets (3.6 × 10⁸ cells/ml) were preincubated with 5 μM or 10 μM honokiol or a solvent control (0.5% DMSO) for 20 min at 37 °C. An aliquot of the supernatant (10 μl) was deposited on a Fuji Dri-Chem slide LDH-PIII (Fuji, Tokyo, Japan), and the absorbance wavelength was read at 540 nm with an ultraviolet-visible spectrophotometer (UV-160; Shimazu, Japan). A maximal value of lactate dehydrogenase (LDH) was observed in the sonicated platelets.

Washed platelets (3.6 × 10⁸ cells/mL) were preincubated with the solvent control (0.1% DMSO) or Ir-11 (10–50 μM) for 20 min at 37 °C. An aliquot of the supernatant (10 µL) was deposited on a Fuji Dri-Chem slide LDH-PIII (Fuji, Tokyo, Japan), and the absorbance wavelength was read at 540 nm using a UV–vis spectrophotometer (UV–160; Shimadzu, Japan). A maximal value of lactate dehydrogenase (LDH) was recorded in the sonicated platelets (Max).

Washed platelets (3.6 × 10⁸ cells/mL) were preincubated with the solvent control (0.1% DMSO) or morin hydrate (40, 80, 100 μM) for 20 min at 37 °C. An aliquot of the supernatant (10 µL) was deposited on a Fuji Dri-Chem slide LDH-PIII (Fuji, Tokyo, Japan) and the absorbance wavelength was read at 540 nm by using an ultraviolet–visible spectrophotometer (UV-160; Shimadzu, Japan). A maximal value of lactate dehydrogenase (LDH) was recorded in the sonicated platelets (Max).

Washed platelets (3.6 × 10⁸ cells/ml) were preincubated with 20, 50, and 100 μM TQ-6 or the solvent control (0.5% DMSO) for 20 min at 37 °C. An aliquot of the supernatant (10 µl) was deposited on a Fuji Dri-Chem slide LDH-PIII (Fuji, Tokyo, Japan), and the absorbance wavelength was read at 540 nm by using an ultraviolet-visible spectrophotometer (UV-160; Shimadzu, Japan). A maximal value of LDH was noted in sonicated platelets.