Ldh kit

Blood samples were collected and centrifuged for 15 minutes at a speed of 2200–2500 RPM. Plasma was pipetted into separate tubes, CK-MB, AST, and LDH levels were analyzed with CK-MB ELISA kit (Senbeijia, Nanjing, China), AST kit (Sigma-Aldrich, St Louis, USA), and LDH kit (Abcam, Cambridge, UK). The manufacturer’s instructions were followed in all test kits used.

The relative levels of LDH in the VL tissue were detected using an LDH kit (Abcam, Germany, ab197000) following the manufacturer’s guidelines. Briefly, 40 mg tissue was homogenized using a T18 digital homogenizer (IKA, USA) at the speed of 2.5 × 10⁴ rpm for 30 s in protein isolation buffer containing 20 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1 μM dithiothreitol (DTT), protease and phosphatase inhibitors and incubated on ice for 10 min. Tissue lysates were subsequently centrifuged for 5 min at 4 °C at 1 × 10⁵ g. Supernatant samples were collected and added to 96-well-plate (50 μL each well). Hereafter, a 50 μL reaction mix supplied by the kit was added to each sample and the fluorescence intensity at 535/587 nm (Ex/Em) was immediately measured using a Tecan multimode microplate reader.

The release of the intracellular enzyme lactate dehydrogenase (LDH), indicative of cell membrane damage, was assessed by a LDH kit (Abcam) according to the manufacturer’s guidelines. Briefly, after cell exposure to the NFC samples, cell culture supernatants were collected and adherent cells were lysated for 30 min with cell lysis buffer diluted in cell culture medium (1:10) and the lysates were collected. Lysates and supernatants were centrifuged at 6800 g during 10 min to avoid any potential interference of NFC. The enzyme activity in the lysates and supernatants samples was measured by reading the absorbance at 450 nm wavelength and reference wavelength 650 nm using a plate reader (Tecan Infinite M200). The experiments were conducted at least three times in duplicate wells for each dose. LDH release (LDH activity in cell culture supernatant) was normalized by the total LDH activity (sum of LDH activity in cell culture supernatants and lysates) which correlates with the total number of cells in order to avoid any underestimation of toxicity [22 (link)].

To investigate the safety of CBEO treatment in mice, the olfactory bulbs of mice were collected, washed with cold PBS, homogenized, and the levels of lactate dehydrogenase (LDH) were determined using an LDH kit in accordance with the manufacturer’s instructions (Abcam, Boston, United States).

To evaluate membrane integrity in the treated HepG2 cells and control, LDH activity was measured using the LDH kit (Abcam, Cambridge, UK). The leakage of LDH from the cells was determined in the medium and the quantification of the produced color was performed at 450 nm using a microplate reader (BioTek ELX800, Winooski, VT, USA). Cells at a density of 2 × 10⁴ cells were cultured with vehicle, Ber, or Ber-NPs for 24 h at the concentrations listed in Table 1. As a result, 10 µL of supernatant was incubated for 30 min with 100 µL of LDH mix (ab65393) and absorbance was measured at 450 nm.