Biotek cytation 5 plate reader

We performed SHERLOCK reactions using 45 nM C2c2 LwaCas13a (GenScript Biotech Corp, United States) resuspended in 1x storage buffer (SB: 50 mM Tris (pH 7.5), 600 mM KCl, 5% glycerol, and 2mM dithiothreitol (DTT)) such that the resuspended protein was at 2.25 μM, 1 U/μL murine RNase Inhibitor (NEB), 10 U/μL T7 RNA polymerase (Lucigen), 136 nM RNaseAlert substrate v2 (ThermoFisher Scientific, United States), 1x SHINE Buffer (SHINE: 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 8.0), 60 mM KCl, and 5% polyethylene glycol (PEG)), and 2 mM of each rNTP (NEB).
We rehydrated the TwistAmp Basic Kit lyophilized pellets (1 pellet per 73.42 μL master mix volume) using the prepared master mix. We added 14 mM MgAOc (TwistDx, United Kingdom) after resuspension to activate the RPA pellets. We then subdivided the master mix for each guide-primer set pair being analyzed, to which was added 22.5 nM gRNA (Integrated DNA Technologies, United States) and 320 nM each of the RPA primers (Integrated DNA Technologies, United States). We prepared SHERLOCK reactions to 70 μL and loaded as 20 μL triplicates into a 384-well plate, with a ratio of 1:5 master mix to sample. We measured fluorescence by the BioTek Cytation 5 plate reader (BioTek, United States) over 3 hours at 37 °C, with readings every 5 minutes (excitation: 485; emission: 528) for quantitative detection.

HMC1, HMC3A, and HMC3B cells were seeded in 24-well plates at 800, 600, and 1400 cells/well, respectively. The next day, media was replaced with 500 μL fresh media containing either vehicle (DMSO) or the indicated concentration of drug (BMS-754807 (Sigma-Aldrich #BM0003), PPP (Santa Cruz #SC-204008), SR10221, SR2595 (Sigma-Aldrich #SML2037), or T0070907 (Fisher #NC1015539); final concentration 1% DMSO). Seven days later, media was removed from all wells and cells were fixed in 10% buffered formalin (Fisher #SF994) for 5 min at room temperature. Wells were washed once in ddH₂O and then stained in 0.05% crystal violet (Sigma-Aldrich #C6158) for 30 min at room temperature. Wells were then washed an additional 3 times in ddH₂O to remove any unbound stain and allowed to dry at room temperature overnight. Once dry, individual wells were imaged at 4X magnification on a BioTek Cytation 5 plate reader (BioTek Instruments, Inc.) and images were stitched using the Gen5 software (v2.09; BioTek Instruments, Inc.) using the default parameters. Colony numbers were quantified manually in ImageJ (Schneider et al., 2012 (link)), where a colony is defined as a cluster of at least 50 individual cells.