Ck kit

The cells were cultured in a 6-well cell culture plate. After differentiation into myotube cells, the cells were pretreated in differentiation medium containing CMP (50/100/200 μmol/L) for 4 h, and the cells were further cultured for 48 h with differentiation medium containing 100 μmol/L hydrogen peroxide and CMP (50/100/200 μmol/L). Then, the supernatant was collected and the contents of antioxidant enzymes CAT and GSH-px in the supernatant of each group were detected with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). CK activity was measured as a differentiation and injury marker using a CK kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Myotoxic activity was quantitatively determined according to the method of Rojas et al. [29 (link)]. Various amounts of venom(0 μg, 5 μg, 10 μg, 15 μg, 20 μg and 25 μg) dissolved in 50 μL of physiological saline (0.9% w/v) were injected intramuscularly (i.m.) into groups of 4 mice in the right gastrocnemius muscle. Control animals received 50 μL of the physiological saline. Three hours after injection, mice were bled from the orbital plexus, under CO₂ anesthesia. Plasma creatine kinase (CK) activity was quantitated by the CK kit (Nanjing Jiancheng Corp. China). CK activity is expressed in units/L, 1 U defined as the amount of enzyme that produces 1 μmol of NADH under the conditions of the assay. The minimum myotoxic dose (MMD) corresponds to the amount of venom that induces an increment of plasma CK activity corresponding to four times the activity of mice injected with physiological saline alone. The challenge doses of venom (3 MMD) was incubated with various amounts of the IgY antibodies (0, 375 μg, 750 μg, 1500 μg and 3000 μg) for 45 min at 37 °C and centrifuged for 5 min. Then, 50 μL of the supernatant was injected subcutaneously into groups of ten mice in in the right gastrocnemius muscle.