Enzymatic Digestion and Manual Dissociation: A Method to Prepare C. elegans Embryos for Cell Culture

Abstract

Source: Sangaletti, R. and Bianchi, L. A Method for Culturing Embryonic C. elegans Cells. J. Vis. Exp. (2013).

This video introduces a method to isolate and sterilely culture embryonic C. elegans cells in vitro.

Protocol

1. Embryonic Cells Dissociation

1. Conduct the next steps of the procedure under sterile conditions using a laminar flow hood. While animals are gown on bacteria plates, the washes and the treatment with the lysis solution containing bleach should eliminate most if not all the bacteria. Thus using a laminar hood at this point of the procedure prevents new contamination of the egg suspension.
2. Resuspend pelleted eggs in 1 ml of 2 mg/ml chitinase (stock in egg buffer pH 6.5) and transfer them to a new sterile 15 ml conical tube. Rock the tube for 10-30 min at room temperature. The exact incubation time changes according to the freshnes....

Results

Figure 1. C. elegans embryos before and after chitinase treatment. (A) Photograph of eggs prior to exposure to chitinase. The arrow points to the transparent and intact eggshell surrounding an embryo. (B)

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
<strong>REAGENTS</strong>
Leibovitz's L-15 Medium (1x) Liquid	Invitrogen	11415-064
Fetal Bovine Serum	Invitrogen	16140-063
Penicillin-streptomycin	Sigma	P4333-100ML
Chitinase from Streptomyces Griseus	Sigma	C6137-25UN
Peanut Lectin	Sigma	L0881-10MG
<strong>EQUIPMENT</strong>
101-1000 &mu;l Blue Graduated Pipet Tips	USA Scentific	1111-2821
10 ml Sterilized Pipet Individually Wrapped	USA Scentific	1071-0810
Ergonomic Variable Volume (100-1000 &mu;l) Pipettor with tip ejector	VWR International Inc.	89079-974
Portable Pipet Aid, Drummond	VWR International Inc.	53498-103
Transfer Plastic Pipet Sterile	VWR International Inc.	14670-114
15 ml Conical Tube	USA Scentific	1475-1611
Sterile 18 gauge Needles	Becton, Dickinson and Co.	305196
Sterile 10 ml Syringes	Becton, Dickinson and Co.	305482
Plastic Syringe Filters Corning 0,20 &mu;m pore size	Corning	431224
Acrodic 25 mm Syringe filter w/5 &mu;m versapor Membrane	VWR International Inc.	28144-095
60x15 mm Petri Dish Sterile	VWR International Inc.	82050-548
100x15 mm Petri Dish Sterile	VWR International Inc.	82050-912
12 mm Diameter Glass Coverslips	VWR International Inc.	48300-560
Clear Cell Culture Plates 24 Well Flat Bottom w/lid	Thomas scientific	6902A09
Dumont #5- Fine Forceps	Fine Science Tools	11254-20
Centrifuge 5702	Eppendorf	22629883
Laminar Flow Hood
Inverted Microscope with x10 objective
Ambient air humidified Incubator