Ldh assay kit

To determine the damage to bacterial cells within the biofilms, an LDH assay was carried out. Briefly, the log phase culture of each test strain (100 µL) with MHB (100 µL) was added into 96-well microtiter plates and incubated for 24 h at 37 °C under static condition. After the incubation period, planktonic cells were discarded by washing thrice with sterile PBS (100 µL). The L. rhamnosus crude biosurfactant (MIC) (100 µL) was then added and the plates were kept for further incubation at 37 °C for 24 h under static conditions. After incubation, the LDH activity was then determined by collecting the supernatant using an LDH assay kit (Sigma-Aldrich^®, Bangalore, India) at 480 nm. The bacterial culture and MHB was used as the negative control.

Following transfection, BEAS-2B cells (1 × 10⁴) were exposed to 75 μg/mL PM2.5 for 48 h. Then the culture supernatants of BEAS-2B cells were collected and LDH release was detected using an LDH assay kit (Sigma-Aldrich) referring to the protocol’s instructions.

Toxicity of HMA was measured via lactate dehydrogenase (LDH) activity using the LDH assay kit (Sigma-Aldrich). Briefly, LDH activity, which is a marker of cell viability, was measured following 8 h co- incubation of the human brain microvascular endothelial cell line with 4–64 μg/ml of HMA. Absorbance was measured at 490 nm for 30 min at 37°C, taking the highest time-point reading before absorbance exceeded the maximum tested standard. LDH activity was calculated using an NADH standard set provided by the kit.

To measure in vitro citotoxicity due to NMDA, released LDH was measured by LDH assay kit (SIGMA) according to manifacture procedure. Data were expressed as % of extracellular LDH activity in treated cells (NMDA or NMDA/CX3CL1) vs. untreated cells.

At the end of each treatment, supernatants were collected and LDH release was quantified using an LDH assay kit (TOX7-Sigma) as per the manufacturer’s instructions. Caspase-1 dependent LDH release that was inhibited by the caspase-1 inhibitor VX-765 (20 μM), was used as a readout for pyroptosis as previously described [31 (link)].