Brdu cell proliferation enzyme linked immunosorbent assay elisa kit

Cell growth was assessed using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). RCC cells (50 μl, 1 × 10⁵ cells/ml) were seeded onto 96-well tissue culture plates. After 24, 48, and 72 h MTT (0.5 mg/ml) was added for an additional 4 h. Subsequently, cells were lysed in a buffer containing 10% SDS in 0.01 M HCl. The plates were incubated overnight at 37°C, 5% CO₂. Absorbance at 550 nm was determined for each well using a microplate ELISA reader. Each experiment was done in triplicate. After subtracting background absorbance, results were expressed as 24 – 72 h cell growth rate calculated in percentage increase compared to controls set at 100%.
Cell proliferation was measured using a BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Calbiochem/Merck Biosciences, Darmstadt, Germany). Tumor cells, seeded onto 96-well microtitre plates, were incubated with 20 μl BrdU-labeling solution per well for 8 h, and then fixed and detected using anti-BrdU mAb according to the manufacturer's instructions. Absorbance was measured at 450 nm.

To investigate apoptotic and necrotic events, the FITC-Annexin V Apoptosis Detection kit (BD Biosciences, Heidelberg, Germany) was used to quantify binding of Annexin V/propidium iodide (PI). After washing tumor cells twice with PBS, 1 × 10⁵ cells were suspended in 500 µL of 1 × binding buffer and incubated with 5 µL Annexin V-FITC and (or) 5 µL PI in the dark for 15 min. Staining was measured by flow cytometer (Fortessa X20, BD Biosciences, Heidelberg, Germany). Ten thousand events were collected from each sample. The percentage of apoptotic and necrotic cells in each quadrant was calculated using DIVA software (BD Biosciences, Heidelberg, Germany). Further analysis was done by FlowJo software (BD Biosciences, Heidelberg, Germany).
A BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Calbiochem/Merck Biosciences, Darmstadt, Germany) was used to evaluate ferroptosis and ROS generation. To evaluate ferroptosis, tumor cells were treated for 48 h with 20, 50, and 100 µM ART or ART combined with 20 µM ferrostatin-1 (Sigma-Aldrich, Darmstadt, Germany), a ferroptosis inhibitor. ROS generation during ferroptosis was verified by treating the RCC cells for 48 h with 20, 50, and 100 µM ART in combination with 0.5 mM Trolox (Sigma-Aldrich, Taufkirchen, Germany), an antioxidant. For more details see “Proliferation” (4.4), as described above.

The FITC-Annexin V Apoptosis Detection kit (BD Biosciences, Heidelberg, Germany) was used to quantify apoptotic and necrotic events. After washing cells twice with PBS, 1 × 10⁵ cells were resuspended in 500 µL of 1 × binding buffer and incubated with 5 µL Annexin V-FITC and/or 5 µL PI in the dark for 15 min. Staining was measured by flow cytometer (Fortessa X20, BD Biosciences, Heidelberg, Germany). The percentage of apoptotic and necrotic cells was calculated using DIVA software (BD Biosciences, Heidelberg, Germany). Further analysis was done by FlowJo software (BD Biosciences, Heidelberg, Germany).
To evaluate ferroptosis, cells were treated with 37.5 µM ART or ART combined with the ferroptosis inhibitor ferrostatin-1 [20 µM] (Sigma-Aldrich, Darmstadt, Germany) for 24 and 48 h. Ferroptosis was assessed using BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Calbiochem/Merck Biosciences, Darmstadt, Germany), as described above. For more details, see Cell Growth and Proliferation (2.4).