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Supplementary Material for Sfarcic et al., 2019

posted on 04.10.2019 by Ivana Sfarcic, Theresa Bui, Erin C Daniels, Emily R Troemel
Supplementary Figure 1. Bioluminescent Signal is Stronger and More Consistent in Lysed C. elegans than in Intact C. elegans.
Bioluminescent signal measured in 100 adult worms that were intact or vortexed with Silicon Carbide beads to obtain worm lysate. Controls did not express NanoLuc but instead expressed a pals-5p::GFP transgene. NanoLuc expressing worms had a single copy insertion of a vha-6p::Nanoluc construct. Signal was measured 10 and 60 minutes after addition of the substrate Furimazine via NanoGlo Reagent (Promega). Red dots represent measurements with an optimal gain across all samples (gain 2662) and grey dots represent measurements with the maximal possible gain to determine the highest detectable luminescent signal for intact worms (max. gain 4095). With a gain of 4095, the signal from worm lysate of vha-6p::Nanoluc worms over-saturated the detection capability of the photomultiplier tube.

Supplementary Figure 2. C. elegans Lysate Can Be Prepared and Measured Directly in Wells of 96-well Plate.
(A, B) Bioluminescent signal measured in worm lysate of (A) pals-5p::Nanoluc or (B) vha-6p::Nanoluc expressing adults. Worm lysate was either prepared in 1.5 ml microfuge tubes or in wells of a 96-well assay plate. For “Lysate from tubes”, 100 ul lysate was prepared in microfuge tubes by vortexing worms, Lysis Buffer and Silicon Carbide beads. Then 50 ul of supernatant was transferred to 96-well assay plates and signal was measured. Additionally, 100 ul of lysate was prepared directly in a well of a 96-well assay plate by vortexing worms, Lysis Buffer and Silicon Carbide beads in a plate sealed with sealing tape. 50 ul of supernatant were transferred to fresh well (“Lysate from wells”) and the remaining lysate with beads was measured in presence of Silicon Carbide beads in grinding well (“Lysate + beads from wells”). Btz = bortezomib.


Article title

Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans

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