C. elegans Blastomere dissectie: een methode om de eierschaal te verwijderen en embryonale cellen te dissocieren

Abstract

Bron: Hsu, C. R., et al. In vitro reconstitutie van ruimtelijke celcontactpatronen met geïsoleerde Caenorhabditis elegans embryoblastomeren en zelfklevende polystyreenparels. J.Vis. Exp. (2019). 

Deze video introduceert een methode om C. elegans blastomeren te isoleren van vroege embryo's.  De resulterende cellen zijn geschikt voor celkweek of ex vivo experimenten.

Protocol

Dit protocol is een fragment uit Hsu et al,   In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyree Beads, J. Vis. Exp. (2019).

1. Isolatie van embryoblastomere
2. Draag handschoenen en een laboratoriumjas om snij- en contact met de bleekoplossing te voorkomen.
   1. Houd elk uiteinde van een microcapillair (capaciteit; 10 μL) met de rechter- en linkerhand vast.
   2. Trek het microcapillai....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aspirator Tube Assembly	Drummond	21-180-13	For the blastomere isolation.
Caenorhabditis elegans strain: N2, wild-type	Caenorhabditis Genetics Center	N2	Strain used in this study
Caenorhabditis elegans strain: KSG5, genotype: zuIs45; itIs37	in house	KSG5	Strain used in this study
Calibrated Mircopipets, 10 µL	Drummond	21-180-13	For the blastomere isolation
CD Lipid Concentrate	Life Technologies	11905031	For the blastomere isolation. Work in the tissue culture hood.
Clorox	Clorox	N. A.	For the blastomere isolation. Open a new bottle when the hypochlorite treatment does not work well.
Coverslip holder	In house	N.A.	For the blastomere isolation.
Dissecting microscope: Zeiss Stemi 508 with M stand. Source of light is built-in LED. Magnification of eye piece is 10X.	Carl Zeiss	Stemi 508	For the blastomere isolation.
Fetal Bovine Serum, Qualified One Shot, Canada origin	Gibco	A3160701	For the blastomere isolation. Work in the tissue culture hood.
General Use and Precision Glide Hypodermic Needles, 25 gauge	BD	14-826AA	For the blastomere isolation
Inulin	Alfa Aesar	AAA1842509	For the blastomere isolation
MEM Vitamin Solution (100x)	Gibco	11120052	For the blastomere isolation.
Multitest Slide 10 Well	MP Biomedicals	ICN6041805	For the blastomere isolation
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140148	For the blastomere isolation.
Polyvinylpyrrolidone	Fisher BioReagents	BP431-100	For the blastomere isolation
Potassium Chloride	Bioshop	POC888	For the blastomere isolation
Schneider’s Drosophila Sterile Medium	Gibco	21720024	For the blastomere isolation. Work in the tissue culture hood.
Sodium Chloride	Bioshop	SOD001	For the blastomere isolation
Sodium Hydroxide Solution, 10 N	Fisher Chemical	SS255-1	For the blastomere isolation
Syringe Filters, PTFE, Non-Sterile	Basix	13100115	For the blastomere isolation.
Tygon S3 Laboratory Tubing,, Formulation E-3603, Inner diameter 3.175 mm	Saint Gobain Performance Plastics	89403-862	For the blastomere isolation.
Tygon S3 Laboratory Tubing,, Formulation E-3603, Inner diameter 6.35 mm	Saint Gobain Performance Plastics	89403-854	For the blastomere isolation.