Lactate dehydrogenase ldh activity assay kit

The cytotoxicity caused by EMS in ALI airway cultures was assessed by measuring LDH release into the basolateral medium using a Lactate Dehydrogenase (LDH) Activity Assay Kit (Roche, Indianapolis, IN). The reaction mixture was freshly prepared by diluting the kit‐supplied catalyst in dye solution at a ratio of 1:45 (v/v) and 100 μL of the reaction mixture were incubated with 100 μL of basolateral medium for 15 min at room temperature in the dark. Fifty μL of stop solution were added to terminate the reactions. Absorbance was measured at 490 nm using a Synergy H4 microplate reader (BioTek, Winooski, VT).

The cytotoxicity of CS was monitored 24 h after 1 (T1) and 12 intermittent exposures (T12), as well as after a 20-day recovery period (PT20) using a Lactate Dehydrogenase (LDH) Activity Assay kit (Roche, Indianapolis, IN). Basolateral media was collected at T1, T12, and PT20 and assayed for LDH release. Equal volumes of the basolateral medium and freshly prepared Reaction Mixture were mixed and allowed to react for 15 min at room temperature in the dark. Reactions were terminated by the Stop Solution. Absorbance at 490 nm was measured using a Synergy H4 microplate reader (BioTek, Winooski, VT).

The cytotoxicity of GA aerosols was assessed using a Lactate Dehydrogenase (LDH) Activity Assay Kit following the procedures recommended by the manufacturer (Roche, Indianapolis, IN, USA). The activity of released LDH was measured in both the apical wash (60 µL) and basolateral medium (100 µL) 24 h following one (T1) and five exposures (T5) as well as after a 7-day recovery (PT7). Apical washes were collected by washing the apical surface of the cultures with PBS (100 µL each wash for a total of two washes). The washes were combined and used for both LDH activity measurement and mucin secretion ELISA (described in the following section). Absorbance at 490 nm was measured using a Synergy H4 microplate reader (BioTek, Winooski, VT, USA).