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. 2024 Apr 5;43(11):2127–2165. doi: 10.1038/s44318-024-00084-7

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© The Author(s) 2024, corrected publication 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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Figure EV1 — (A) Table of medium volumes used in this study and the corresponding medium heights of both top and bottom menisci. Images of each plate-type containing different medium volumes were used to measure menisci heights. Known well diameters were used to convert menisci heights from pixels to mm. ‘High’ refers to the standard culture volumes used. (B) Representative trace of fluorescence intensity indicative of pericellular oxygen concentrations under different medium volumes, measured for 24 h in 96-well plates. The bar graph shows relative oxygen levels taken at 16 h (arrow in representative trace) (n = 4 biological replicates). AA antimycin A. (C) Western blot of mitochondrial respiratory complexes I–V after 16 h of medium volume change in 12-well plates (n = 3 biological replicates). (D) Oxygen consumption rate (OCR) of permeabilised 3T3-L1 adipocytes upon different substrate stimulation after 16 h of medium volume change (n = 5 biological replicates). (E) Extracellular medium glucose and lactate measurements in 24-well or Lumox plates after 16 h culture in high or low medium volumes (n = 3 biological replicates). (F) Medium glucose concentration after 16 h of medium volume change in 12-well plates (n = 6 biological replicates). (G) Extracellular medium glucose and lactate measurements after 16 h medium volume change with different starting glucose concentrations in 12-well plates. (n = 4 biological replicates). (H) OCR measurements from different cell densities in 96-well plates (n = 3 biological replicates). (I) Extracellular medium glucose and lactate measurements after 16 h medium volume change with different cell densities in 12-well plates measured by DNA concentration. (n = 3 biological replicates). Data information: Data were represented as mean ± SEM (B, D–I). ****p < 0.0001 by two-way ANOVA with Šidák correction for multiple comparisons (E).