Ultrafree cl centrifugal filter units

The synchronized population of the ∼2,000 L1 stage worms was placed on 100 mm NGM agar plates seeded with E. coli OP50 bacteria and supplemented with or without 50 µM Cd. After 48 h, young adult hermaphrodites were collected from plates with M9 buffer and washed free from E. coli OP50 by two rounds of centrifugation (3,500 × g for 2 min). Worms, resuspended in M9 buffer were concentrated using Ultrafree-Cl Centrifugal Filter Units (MILLIPORE). Total RNA was isolated from worms with TRIZOL reagent (Invitrogen) according to the manufacturer’s recommendations. gDNA was cleared from the RNA preparations by DNAse (Roche) prior to the first strand cDNA synthesis. RT-qPCR was carried out as described earlier (Gayomba et al., 2013 (link)). Data were normalized to the expression of actin, act-1. The fold-difference (2^−ΔΔCq) or relative quantities were calculated using the CFX Manager Software, version 1.5 (BioRad).

The synchronized population of the L1 stage worms were grown on NGM plates to adult stage. Synchronized young adult worms were collected and transferred to the 60 mm NGM plates seeded with OP50 and supplemented with or without 50 µM CdCl₂. After 24 h, worms were collected and washed with excess S-basal (0.1M NaCl, 0.05 M KH₂PO₄, 5 mg/mL cholesterol) 3 times. Each time, worms were incubated in S-basal for 15 min before centrifugation to remove bacteria from the worm gut. Worms were finally filtered out with Ultrafree-Cl Centrifugal Filter Units (MILLIPORE) at 30 × g for 1 min to remove the remaining bacteria. Worms were then anesthetized with ice-cold 0.2% (w/v) NaN₃ for 2 min. After all the worms were completely immobilized, they were washed twice with the de-icing agent, ice-cold 1.5% (w/v) CH₃COONH₄. Immobilized animals were transferred to 200 nm thick silicon nitrite window (SiMPore, Inc.) and straightened using an eyelash. Excess liquid was removed using fine-tapered paper wick (MiTeGen, United States). Then the window with attached worms was plunge-frozen in liquid nitrogen slush and dried overnight using a Balzers High Pressure Freezer. Freeze-dried worms were stored in a cryovial at room temperature with desiccants.