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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
From:
Curr Protoc Immunol. Author manuscript; available in PMC 2017 April 1.
Published in final edited form as:
Curr Protoc Immunol. 2016; 113: 3.16B.1–3.16B.14.
Published online 2016 April 1. doi: 10.1002/0471142735.im0316bs113

Table 1

Troubleshooting Guide for Measuring Bioenergetics with an Extracellular Flux Analyzer
ProblemComments and Suggestions
Poor basal signal
 - Low cell numberIncrease the number of cells per well.
 - Low cell viabilityKeep cells on ice for all steps before plating and/or reduce processing time.
High variation between replicates
 - Uneven cell number platedMake sure the correct volume of cells is added in each well of the cell plate, and that the volume of cells is maximal 100 μl.
 - A certain probe does not detect signals properlyMake sure all probes are hydrated for 4 – 24 hours with sufficient volume of Calibrant XF, and use cartridges before the expiration date.
 - Cell monolayer was disturbed during XF media additionMake sure the cell plate is PDL-coated and the plate has been spun down after adding the cells. Using too many cells, as well as having
substantial numbers of dead cells in the well, results in multiple cell layers, and will increase disturbance of the cells after injection.
High variation between repeated measurements within a loop
 - Decline in basal measurementsMake sure to keep the cell plate in a 37°C non-CO2 incubator for 30–60 minutes before putting the cells in the machine.
 - Decline in measurements after drug injectionsThis is sometimes, but not exclusively, seen after FCCP injection. The drug concentration may not be optimal. Titration of the drug might eliminate this problem, however, other biological factors, independent of drug dose, could also contribute to a drop.
 - Cell monolayer was disturbed after injectionMake sure the cell plate is PDL-coated and the plate is spun down after plating the cells. Using too many cells, as well as having substantial numbers of dead cells in the well, results in multiple cell layers, and will increase disturbance of the cells after injection.
Minimal or unexpected changes in OCR after drug injection
 - No drug is injectedMake sure all drugs are added directly into the correct ports in the right orientation without contaminating other ports.
 - Inconsistent injection of drugs between wellsFill all injection ports that will be injected with drugs of interest. Make sure all injection ports that will be injected are filled with the proper volume to ensure balanced injection.
 - Concentration of drug is too lowTitration of drug concentration may be required.
 - Incorrect orientation of cartridge or cell plate during plating or runningThe drugs will be injected in a wrong order if the orientation of the cartridge is incorrect. Ensure correct orientation (see user's manual of the EFA) of the cell plate and the cartridge, both during plating/drug loading and during the run.