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

Protokol

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 freshness of the enzyme and the temperature of the room and should therefore be determined for each preparation. It is recommended to start monitoring the eggs under an inverted cell-culture microscope after 10 min of incubation. Note: in our experience, low pH increases the chitinase enzymatic activity. For this reason we use egg buffer at pH 6.5 to dissolve chitinase (recipe reported above, where pH is adjusted to 6.5 using NaOH).
3. When ~ 80% of the eggshells are digested by the chitinase treatment (Figures 1 A-B), pellet the eggs by centrifugation at 900 x g (~2,500 rpm) for 3 min. Using a P1000 pipettor and sterile tips, remove carefully the supernatant and add 3 ml of L-15 medium.
4. Transfer the eggs into a 6 cm diameter plate and gently dissociate the cells using a 10 ml sterile syringe equipped with a 18 G needle. Monitor the degree of dissociation by placing a drop of suspension into a fresh plastic Petri dish and by viewing under the microscope. Do not aspirate air into the syringe during this procedure to avoid damaging cells. Continue the dissociation until ~ 80% of the cells are dissociated.
5. Filter the suspension using a sterile 5 μm Millipore filter. Cell suspensions must be filtered in order to remove cell clumps, undigested eggs and hatched larvae. Filter additional 4-5 ml of fresh L-15 media through the filter to recover all the cells. Do not use excessive force during the filtration step to avoid damaging the filter and/or the cells.

2. Culturing Cells

1. Pellet the dissociated cells by centrifugation at 900 x g (~2,500 rpm) for 3 min. Using a P1000 pipettor and sterile tips carefully remove all the supernatant. Resuspend the pelleted cells in complete L-15 medium and plate 1 ml/well. The amount of the medium added depends on the number of 8P plates used, the confluence of the worms on the plates, and the type of experiments that will be performed on the cells. The cell density can be determined using a hemocytometer. For patch-clamp recordings plating density of ~ 230,000 cells/cm² is optimal.
2. Keep the 24 wells plate in a plastic Tupperware container containing wet paper towels to avoid evaporation of culture medium. Store the container in a humidified incubator at 20 °C and ambient air.
3. The cells are usually ready for the experiments within 24 hr when the morphological differentiation and expression of GFP markers are complete. Cells can be kept in culture for up to 2 weeks but they are usually most healthy up to 7-9 days after plating. The medium needs to be replaced once a day to maintain healthy cells.

Sonuçlar

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) Eggs treated with chitinase for 10 min. The eggshells have been digested and are no longer visible around th...

Malzemeler

Name	Company	Catalog Number	Comments
REAGENTS
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
EQUIPMENT
101-1000 μl Blue Graduated Pipet Tips	USA Scentific	1111-2821
10 ml Sterilized Pipet Individually Wrapped	USA Scentific	1071-0810
Ergonomic Variable Volume (100-1000 μl) 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 μm pore size	Corning	431224
Acrodic 25 mm Syringe filter w/5 μm 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