Zdrazilova Lucie, Hansikova Hana, Gnaiger Erich
Department of Pediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General Hospital in Prague, Prague, Czechia.
Oroboros Instruments, Innsbruck, Austria.
PLoS One. 2022 Mar 3;17(3):e0264496. doi: 10.1371/journal.pone.0264496. eCollection 2022.
Measurement of oxygen consumption of cultured cells is widely used for diagnosis of mitochondrial diseases, drug testing, biotechnology, and toxicology. Fibroblasts are cultured in monolayers, but physiological measurements are carried out in suspended or attached cells. We address the question whether respiration differs in attached versus suspended cells using multiwell respirometry (Agilent Seahorse XF24) and high-resolution respirometry (Oroboros O2k), respectively. Respiration of human dermal fibroblasts measured in culture medium was baseline-corrected for residual oxygen consumption and expressed as oxygen flow per cell. No differences were observed between attached and suspended cells in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. The electron transfer capacity was higher in the O2k than in the XF24. This could be explained by a limitation to two uncoupler titrations in the XF24 which led to an underestimation compared to multiple titration steps in the O2k. A quantitative evaluation of respiration measured via different platforms revealed that short-term suspension of fibroblasts did not affect respiratory activity and coupling control. Evaluation of results obtained by different platforms provides a test for reproducibility beyond repeatability. Repeatability and reproducibility are required for building a validated respirometric database.
培养细胞耗氧量的测量广泛应用于线粒体疾病诊断、药物测试、生物技术和毒理学领域。成纤维细胞以单层形式培养，但生理测量是在悬浮或贴壁细胞中进行的。我们分别使用多孔板呼吸测定法（安捷伦Seahorse XF24）和高分辨率呼吸测定法（奥罗波若斯O2k）来探讨贴壁细胞与悬浮细胞的呼吸是否存在差异。在培养基中测量的人皮肤成纤维细胞呼吸，针对残余氧消耗进行了基线校正，并表示为每个细胞的氧流量。在活细胞的常规呼吸以及用寡霉素抑制ATP合酶后获得的泄漏呼吸方面，贴壁细胞和悬浮细胞之间未观察到差异。O2k中的电子传递能力高于XF24。这可以通过XF24中仅进行两次解偶联剂滴定的限制来解释，与O2k中的多次滴定步骤相比，这导致了低估。通过不同平台测量的呼吸的定量评估表明，成纤维细胞的短期悬浮不会影响呼吸活性和偶联控制。对不同平台获得的结果进行评估，为超越重复性的可重复性提供了检验。建立经过验证的呼吸测定数据库需要重复性和可重复性。
