Cytochrome c elisa kit

Vehicle- or compound-treated Mf were lysed with RIPA buffer (Himedia Laboratories Pvt Ltd., Maharashtra, India) for 1 h in the presence of protease inhibitors. The Mf lysates were centrifuged at 300× g for 3 min at 4 °C to remove cell debris, and the supernatants were centrifuged at 16,000× g for 20 min at 4 °C to pellet mitochondria and obtain a post-mitochondrial supernatant fraction.
The cytochrome c ELISA kit (Invitrogen, Maharashtra, India) was used to estimate cytochrome c protein content in the post-mitochondrial supernatant fraction as per the manufacturer’s instructions. Measurements were performed in duplicate, and the cytochrome c content was analyzed at 450 nm.

The release of cytochrome C from mitochondria was determined using the Cytochrome C ELISA Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions [18 (link)]. LoVo cells were seeded at 5 × 10³ cells/well and incubated in a medium containing 5 µM 5-FU for 48 or 72 h. Next, the medium was removed and incubated with a cell lysis buffer on ice for 10 min. Then, the lysates were centrifuged at 10,000 × g for 5 min; a reaction buffer was added to each well and incubated for 3 h at room temperature. Next, biotin-conjugated, diluted streptavidin–horseradish peroxidase and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added to all the wells. Finally, the absorbance was read at 600 nm using an automatic spectrophotometer PowerWave X (Bio-Tek, Santa Clara, CA, USA).

Levels of cytochrome C in the cell cytoplasm of A549 cells forming ALI multilayered co-cultures were quantified by Cytochrome c ELISA Kit (Invitrogen, Biosciences Ltd., Ireland), as previously described [19 (link)].

Quantification of cytochrome c was carried out using cytochrome c ELISA kit (Invitrogen, USA) according to the manufacturer's instructions. Briefly, 1 × 10⁵ cells/mL of H400 cells were seeded and treated with DC extract, fractions DCc and DCd, and subfractions DCc15 and DCd16 of D. cinnabari at concentration of IC₅₀ for 24 hours. Untreated cells were used as negative control. After 24 hours of treatment, cells were harvested and washed twice with ice-cold PBS. The resulting cell pellets were stored at −80°C until they were used. Cell proteins were extracted using RIPA lyses buffer (Bio Basic Canada INC, Canada) supplemented with protease inhibitor, phosphatase inhibitor, and phenylmethylsulfonyl fluoride (PMSF) according to manufacturer's instructions. Cytochrome c in cell extracts was quantified using ELISA kit. Cytochrome c at concentration of 0–5 ng/mL was used as standard. Absorbance reading at 450 nm for samples and standard was measured using Tecan Infinite M200 Pro ELISA plate reader (Männedorf, Switzerland). Sample concentrations were determined using cytochrome c standard curve and expressed as ng per mL.

The cytochrome c ELISA kit (Cat # KH01051, Thermo Fisher Scientific, Braamfontein, JHB, SA) was used to detect and quantify the level of cytosolic cytochrome c expression. The kit is provided with an affinity tag antibody and a reporter conjugated detector complex (capture antibody/analyte/detector antibody) immobilised on the 96-well plates. The treated MCF-7/DOX cells used as samples were prepared by centrifuging the cells at 500× g for 5 min at 4 °C. The cells were lysed in cell extraction buffer provided by the kit for 30 min on ice. After centrifugation at 13,000× g for 10 min, the supernatant was collected and used as a sample. The assay was performed by mixing equal volume (100 µL) of samples and biotin conjugate solution added on a 96-well plate and incubated for 1 h at room temperature on a plate shaker set at 400× g. After that, each well was washed three times with 350 µL of 1× wash buffer (provided in the kit), and 100 µL of 1× streptavidin-horseradish peroxidase (HRP) solution was added and incubated for 30 min. The plate was washed with 1× wash solution, 100 µL of stabilised chromogen was added, and the mixture was incubated for 30 min in the dark. Next, 100 µL of stop solution (provided in the kit) was added to each well before 1 min shaking and an optical density reading at 450 nm (Perkin-Elmer, Victor Nino^TM multimode plate reader, Pontyclun, UK).