Enzymatic Digestion and Manual Dissociation: A Method to Prepare C. elegans Embryos for Cell Culture
Published: April 30, 2023
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 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. Resuspend pelleted eggs in 1 ml of 2 mg/ml chitinase (stock in egg buffer pH 6.5) and transfer …
Representative 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) Eggs treated with chitinase for 10 min. The eggshells have been digested and are no longer visible around th…
Materials
| 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 |
| 60×15 mm Petri Dish Sterile | VWR International Inc. | 82050-548 |
| 100×15 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 |
Cite This Article
Enzymatic Digestion and Manual Dissociation: A Method to Prepare C. elegans Embryos for Cell Culture. J. Vis. Exp. (Pending Publication), e20159, doi: (2023).