Multicolor plate reader fluorescence calibration

Abstract

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

A modular cloning toolbox including CRISPRi for the engineering of the human fungal pathogen and biotechnology hostCandida glabrata

“…All arbitrary fluorescence values were normalized to the calibrant dye sulforhodamine-101 (Supplementary Figure 7) . 39 Similar to the green fluorescence, also for the red fluorescence vector version 1.1 showed higher expression than 2.2 (between 11 and 2-fold difference depending on promoter (Supplementary Figure 7A, B and D) . Vector version 1.1 yielded a 44-fold expression over background with the strongest promoter tested (the medium-level TDH3 promoter) and vector version 2.2 a 4-fold expression level over background for the same promoter.…”

“…29 Arbitrary fluorescence units were then normalized to the chemical calibrant fluorescein to facilitate lab-to-lab portability of the data. 39 Figures 2A and B show that all 19 YTK promoters were functional in C. glabrata , showing at least 3-fold expression over background in the higher expression vector version 1.1 (Figure 2A) . The background was defined as the autofluorescence of C. glabrata cells not harboring a plasmid.…”

“…Venus and mRuby2 were used as read-outs for protein expression levels from different constructs and fluorescence was calibrated using previously described calibrant dyes for green and red fluorescence. 39…”

“…Calibrating arbitrary fluorescence units into nM calibrant dye fluorescence was performed as described before. 39 Linear regression was used to derive the conversion factor.…”

“…Venus and mRuby2 were used as read-outs for protein expression levels from different constructs and fluorescence was calibrated using previously described calibrant dyes for green and red fluorescence. 39 All experiments were performed in C. glabrata ATCC 2001 HTL -. 38 This strain is a clinical isolate frequently used for C. glabrata research including the creation of the C. glabrata deletion collection 38 .…”