A Multiwell Liquid-Based Assay for Screening C. elegans Knockdowns

Samenvatting

Source: Anderson, Q. L. et al. A High-throughput, High-content, Liquid-based C. elegans Pathosystem. J. Vis. Exp. (2018)

This video demonstrates a high-throughput screening method using Caenorhabditis elegans to study gene knockdowns during host-pathogen interactions. It outlines the procedure for preparing knockdown worms and transferring them to assay plates to evaluate the effects of pathogen-induced worm killing.

Protocol

1. Adaptation for Screening Multiple Caenorhabditis elegans Strains or Knockdowns (Ribonucleic Acid Interference (RNAi) Screen Setup)

Note: This is an RNAi screen described for a 24-well plate setup.

1. Add 1 mL of S Basal per well of a 24-well plate containing worms seeded with RNAi. Gently agitate worms by shaking the plate, then transfer worms to an empty, sterile 24 deep-well plate. Allow worms to settle gravitationally (~5 minutes).
2. Aspirate supernatant, leaving approximately 1 mL. Add 7 mL of S Basal to each well.
3. Repeat steps 1.1 - 1.2 a minimum of 2 more times. After final wash, aspirate supernatant, leaving approximately 400 µL.
4. Using the Resampler function of the worm sorter, sort 22 worms from the 24-well plate worm suspension into each well of a 384-well plate (each well of a 24-well plate yields 8 - 12 wells of a 384-well plate).
   1. To avoid starvation, pipette bacteria into 384-well plates (either a strain that serves solely as food, such as OP50, or a pathogenic strain) prior to sorting.
5. After worms have been sorted into the 384-well plate, add small molecules or other experiment-specific materials.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
COPAS FP BioSorter	Union Biometrica		Large object flow cytometer/worm sorter
24 Deep-Well RB Block	Thermo Fisher Scientific	CS15124
384-Well plate	Greiner Bio-One	MPG-781091
Nematode Growth Media (NGM)			Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL Calcium chloride (1 M), 1 mL Magnesium sulfate (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water
S Basal			Amount per liter: 5.85 grams NaCl, 6 grams Monobasic potassium phosphate, 1 gram Dibasic potassium phosphate, and 1 Liter of milli-Q water
Agar	USBiological Life Sciences	A0930
NaCl	USBiological Life Sciences	S5000
Peptone	USBiological Life Sciences	P3300
Calcium chloride	USBiological Life Sciences
Magnesium sulfate	Fisher Scientific	M63-500
Phospate buffer			Amount per liter: 132 mL of Dibasic potassium phosphate (1M) and 868 mL of Monobasic potassium phosphate (1M)
Monobasic potassium phosphate	Acros Organics	7778-77-0
Dibasic potassium phosphate	USBiological Life Sciences	P5100
Bacterial Strains
P. aeruginosa (PA14)
E. coli superfood (OP50)
E. coli RNAi expressing bacteria (HT115)
Worm Strain
glp-4(bn2) (Beanan and Strome, 1992, PMID: 1289064)