Sep 23, 2022
Embryo stage C. elegans dissociation for FACS isolation and RNA-seq analysis of intestine-specific cells
- 1Colorado State University, Fort Collins
Protocol Citation: Robert TP Williams, Erin Osborne Nishimura 2022. Embryo stage C. elegans dissociation for FACS isolation and RNA-seq analysis of intestine-specific cells. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbw9plzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 21, 2022
Last Modified: September 23, 2022
Protocol Integer ID: 57237
Keywords: C. elegans, FACS, single cell suspension, cell dissociaiton, embryo stage, intestine
Funders Acknowledgement:
National Institutes of Health
Grant ID: R35GM124877
Bridge to Doctorate at Colorado State University
Grant ID: 1612513
National Science Foundation
Grant ID: 2143849
Webb-Waring Biomedical Research Award
Grant ID: E.O.N.
Abstract
This protocol is for generating a single cell suspension suitable for isolation of intestine-specific cells through Fluorescence Activated Cell Sorting (FACS) from embryo stage C. elegans. This protocol utilizes treatment with Chitinase and Pronase E to disrupt the cuticle. Embryos are mechanically homogenized with 21G syringe needle.
Materials
Strains:
- FACS control C. elegans strain, i.e. N2
- FACS sorting C. elegans strain, i.e. JM149 caIs71[elt-2p::GFP::HIS-2B::unc-54 3'UTR + rol-6(su1006)]
Reagents:
L15-10 solution
- Leibovitz's L-15 Medium (Thermo 21083027)
- Fetal Bovine Serum (heat inactivated) (Thermo 10438026)
- 100X Penicillin Streptomycin solution (Thermo 15140148)
- Sucrose powder
Stock solutions for egg buffer
- 2M NaCl
- 2M KCl
- 1M CaCl2
- 1M MgCl2
- 1M HEPES pH 7.2
Enzymes
- Chitinase from Streptomyces griseus (Sigma C6137-5UN)
- Pronase E, Protease from Streptomyces griseus (Sigma P8811-1G)
Consumables:
- standard 1.5 ml tubes
- Stericup 0.2 micron filter (Fisher S2GPU05RE)
- 21 guage 1 inch needle (fisher 14-826C)
- 1 ml syringe (fisher 14-823-30)
- 35-micron nylon mesh filter caps (Stellar Scientific FSC-FLTCP)
- 5 ml sterile polypropylene round-bottom tube (STEMCELL Technologies 38057)
- Bio-Rad TC20 automated cell counting slide (Bio-rad 1450011)
Equipment:
- Fixed angle rotor centrifuge (Eppendorf 5424)
- Swinging bucket rotor refrigerated centrifuge (eppendorf 5810R)
- 15 ml tube and 1.5 ml tube adapter (eppendorf 022638704, eppendorf 022638742)
- Fluorescent microscope
- Nutating mixer
- Bio-Rad TC20 automated cell counter
Before beginning
Before beginning
Prepare reagents in advance
L15-10 Buffer: Mix 500 ml Leibovitz's L-15 Medium, 50 ml Fetal Bovine Serum (heat inactivated), 50 ul of 100x Penicillin-Streptomycin solution and 7.7 g sucrose. Filter with 0.2 micron pore filter. Store at 4ºC.
Egg Buffer: Mix 29.5 ml of 2M NaCl, 12 ml of 2M KCl, 1 ml of 1M CaCl2, 1 ml of MgCl2, 12.5 ml of 1M HEPES-NaOH pH 7.2 and 435 ml molecular grade water. Filter with 0.2 micron pore filter. Store at 4ºC.
Chitinase solution (1 U/ml): Dissolve 5 units of Chitinase from Streptomyces griseus (Sigma C6137-5UN) in 5 ml of Egg Buffer. Nutate the solution for approximately 10 minutes until dissolved. Prepare 1 ml aliquots in 1.5 ml tubes. Store aliquots at -20ºC.
Pronase E solution (15 mg/ml): Weigh 150 mg of Protease from Streptomyces griseus (Sigma P8811-1G) into a 15 ml tube. Dissolve the enzyme in 10 ml of Egg Buffer. Nutate the solution for approximately 10 minutes until dissolved. Prepare 1 ml aliquots in 1.5 ml tubes. Store aliquots at -20ºC.
On day of protocol:
Cool swinging bucket centrifuge to 4ºC
Thaw pronase and chitinase aliquots at room temperature
Place L15-10 and egg buffer on ice
Starting material:
Strains: N2, fluorescent sorting strain
Perform this protocol on both strains in parallel
Note: The volumes for enzymatic treatments in this protocol require an embryo pellet less than 200 uL. If embryo pellet exceeds 200 ul, utilize 2x the embryo pellet volume.
Chitinase Treatment
Chitinase Treatment
Centrifuge embryo suspension at 2000 rcf for 1 minute in swinging bucket centrifuge
Resuspend the embryo pellet in 1 ml of M9 and transfer to a 1.5 ml tube.
Pellet the embryos at 2000 rcf for 1 minute in a centrifuge
Aspirate and discard the supernatant
Transfer 10 ul of embryo pellet to 1ml of Qiazol and store at -80°C for downstream RNA analysis
Resuspend the embryo pellet from Step 7 in 0.5 ml egg buffer and 1 ml chitinase (1 U/ml)
Incubate for 20 min rotating/nutating at room temperature
Verify eggshell digestion by visualizing the chitinase treated embryos under a microscope. Early embryos should change shape, and pretzel stage embryos should release from their eggshell.
Pellet the embryos at 200 rcf for 5 min in fixed angle rotor centrifuge
Aspirate and discard the supernatant
Pronase treatment and dissociation
Pronase treatment and dissociation
Resuspend the chitinase treated embryo pellet in 200 ul pronase (15 mg/ml) and 500 ul egg buffer
Attach a 21 guage 1¼ inch needle to a sterile 1 ml syringe
Disrupt the embryo vitelline membrane and release cells by passing the embryo suspension through the needle 100 times, generating a worm slurry
Visually confirm embryo dissociation by viewing a 2 ul sample of worm slurry on a fluorescent microscope
Quench the pronase treatment by adding 800 ul of L15-10 media to worm slurry
Store the sample on ice until all strains are completed
Wash and harvest single cells
Wash and harvest single cells
Wash away excess pronase from the worm slurry
Pellet the worm slurry at 500 rcf for 5 mins in swinging bucket centrifuge cooled to 4ºC
Aspirate and discard the supernatant
Resuspend the worm slurry in 1 ml of L15-10 media.
Pellet the worm slurry at 500 rcf for 5 mins in swinging bucket centrifuge cooled to 4ºC
Aspirate and discard the supernatant
Resuspend the worm slurry in 1 ml of L15-10 media.
Harvest the cells
Pellet undissociated embryos at 100 rcf for 1 minute in swinging bucket centrifuge cooled to 4ºC
NOTES:
- This step will separate the dissociated cells from intact embryos
- Cells will remain in the supernatant
- Ensure your cell type of interest is not lost during this step.
- Visually confirm fluorescent cells are present in the supernatant.
- Visually confirm fluorescent cells are not present in the pellet.
- You may need to reduce the centrifuge speed and/or time if fluorescent cells are in the pellet of this step.
Aspirate 1 ml of the cell-containing supernatant. Keep the pipette away from the pelleted worm debris.
Dispense the cell suspension though a 35-micron nylon mesh filter into a 5 ml flow cytometry tube
Pellet undissociated embryos at 100 rcf for 1 minute in swinging bucket centrifuge cooled to 4ºC
Perform an additional round of cell harvest for the sorting strain only (Step 21)
Total cell suspension volumes:
- Control strain = 1ml
- Sorting strain = 2ml
- Transfer 70 ul of cells to 1 ml of Qiazol and store at -80°C for downstream RNA analysis
- Continue to step 24
- Retain the remaining ~2ml of cells for FACS
Measure approximate cell concentration
Measure approximate cell concentration
Load 10 ul of cell suspension to a Bio-Rad TC20 automated cell counting slide
This protocol should yield between to total cells
Dilute the sample to if above this concentration with L15-10
Microscopically confirm fluorescent cells are present in the cell suspension
Move on to FACS protocol