Telotaggg telomerase pcr elisa

As an indicator of cell culture longevity, telomerase activity was assessed using Telo TAGGG Telomerase PCR ELISA (Roche, Germany) following manufacturer’s protocols. In brief, RTgutF cells (20,000 cells) were harvested at different passages, lysed and telomeric repeat amplification was performed with provided substrate primers (20 min elongation, 5 min inactivation and 30× amplification cycles). Products were denatured and hybridised with Digoxigenin (DIG), followed by immobilisation with biotin and streptavidin coating. Samples were semi-quantitatively assessed using the internal standard and horseradish peroxidase (Anti-DIG-HRP). The limit of detection was considered as the two-fold background activity. Protein content was assessed by bicinchoninic acid protein assay kit (Pierce, USA) following the instructions of the manufacturer. Relative telomerase activity was normalized to 1 mg/mL total protein.

To test the effects of the AuNP treatment compounds on telomerase activity in IMR90, cells were treated with each test compound for 15 passages as described above. The Telo TAGGG Telomerase PCR–ELISA (Roche Applied Science, Penzberg, Germany) was performed on the samples collected at P2, P5, P9, P11, P13, and P15 to investigate the telomerase activity profile over time. Telomerase activity in IMR90 cells was measured in a two-part procedure; the first part is a PCR-based method, and the second part is an ELISA per the PCR–ELISA kit instructions, as described previously [40 ].
To determine the effects of the AuNP treatment compounds on telomerase activity in HDFn, cells were plated as described, and telomerase activity was measured by Q-TRAP [46 (link),47 (link),48 (link)]. Telomerase activity was determined in whole cell lysates by Q-TRAP in HDFn as described by Samuel, et al., 2022 [30 (link)]. Cells were collected after 6, 12, 24, and 72 h of treatment at the concentrations in Table 2, under standard culture conditions. All samples were run in triplicates.

Total kidney protein was used to assess telomerase activity with a commercial kit (TeloTAGGG telomerase PCR ELISA; Roche), which combines PCR and enzyme-linked immunosorbent assay techniques (Additional file 1: Figure S1). To determine the intensity of telomerase activity, we assessed luminescence in a microplate reader (Thermo Fisher Scientific).