Fig. 1: A bioluminescence screening protocol for quantification of luminal cargo proteins in EVs.
From: Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles
a Selection criteria and overview of EV-sorting protein candidates. The red solid lines indicate the 25%, 50% and 75% percentile values. b SEC elution profiles of conditioned media from HEK-293T cells expressing Tluc or CD63-Tluc. Tluc activity in each fraction was quantified directly (group PBS) or after membrane lysis (group Triton) and normalized to the fraction with the highest signal. EVs and soluble proteins were recovered in fractions 0-3 and 4–12, respectively. c Scheme of differentiating Tluc forms in conditioned media. d Percentage of intravesicular Tluc for CD63-Tluc using fractionated and unfractionated media. Results are shown as the mean ± standard deviation of three biological replicates. Two-sided Student’s t test (P > 0.9999). ns: not significant. e Outline of the screening procedure and data analyses. HEK-293T cells were grown in 96-well microplates and co-transfected with Tluc fusion plasmid and Nluc plasmid. Cell cultures were centrifuged and Tluc activity was measured in the cell pellet and conditioned media. Nluc activity was only quantified in the conditioned media. c, e Created with BioRender.com. Source data are provided as a Source Data file. SEC size exclusion chromatography.
