Figure 4.

SMase D binding and cell death of human keratinocytes induced by Loxosceles venom. HaCaT cells (5 × 10⁴/well) were treated with 10 μg of L. laeta venom, preincubated or not with each inhibitor to evaluate cell death, binding of the SMase D to the keratinocyte cell membrane and secretion of MMP-2 and MMP-9. Cell death was evaluated after 72 h of incubation by the MTT method. The ability of the toxins to bind to the surface of keratinocytes was analysed by flow cytometry using a horse polyclonal serum against L. intermedia SMases D. Detection of MMP-2 and 9 was performed using Calbiochem MMP- 2 and 9 ELISA Kits. Data are represented as mean ± SEM of duplicates representative of three independent experiments. Statistically analysed by One-Way ANOVA followed by Tukey HSD test. (*)Significant difference in relation to the buffer (p < .05); (#) Significant difference in relation to the venom (p < .05).