Ldh cytotoxicity kit

Bone-marrow derived macrophages (BMDM) were obtained from BALB/c mice femurs and cultivated at 37°C under 5% CO₂ for 7 days in RPMI medium supplemented with 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine and 30% L929 cell culture supernatant (a source of macrophage colony-stimulating factor). Next, differentiated BMDM were harvested using cold saline solution. BMDM (1 × 10⁵/well) were plated on 96-well plates and cultured at 37°C under 5% CO₂ in RPMI medium without phenol red, supplemented with 1% Nutridoma™-SP (instead of FBS) for 24 h. BMDM were then treated with Brazilian green propolis (EPP-AF^®) at variable concentrations (3.12–200 μg/mL, serial dilution factor 1:2) at 37°C for 48 h. Next, the cell supernatants were collected and LDH activity was quantified using the LDH cytotoxicity kit (Roche), while BMDM were incubated for an additional 4 h in culture medium containing 10% v/v sodium resazurin salt. Uninfected BMDM cultures supplemented with RPMI medium (Medium) or Triton X-100 (2% v/v) were used as cell viability and death controls, respectively. Absorbance was read at 570 nm and 600 nm for the resazurin sodium assay and at 492 nm for the LDH assay by spectrophotometer (SPECTRA Max 190).

Damage induction by C. albicans in TR146 cells was performed as described [70 (link)]. Briefly, monolayers of TR146 cells in 96-well tissue culture plates prepared as described above were infected with2 x 10⁴ yeast cells per well and incubated for 24 hours at 37°C and 5% CO₂. Control wells were incubated with medium only or with 1% Triton-X-100 to determine 100% damage. LDH release into the supernatant was quantified with the LDH cytotoxicity kit (Roche) according to the manufacturer’s instructions. For assessing LDH concentrations in the supernatant of reconstructed human epidermis, a standard curve was generated with recombinant LDH enzyme.