Isolation of Neurons from Caenorhabditis elegans via Fluorescence-Activated Cell Sorting

Samenvatting

Source: Germany, E. M., et al. Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans. J. Vis. Exp. (2019)

This video demonstrates the isolation of fluorescently labeled neurons from Caenorhabditis elegans. A cell suspension is obtained from transgenic worms engineered to express fluorescent protein-fused vesicular transmembrane transporters in cholinergic neurons. The fluorescently labeled neurons are then isolated using fluorescence-activated cell sorting (FACS) and maintained in culture.

Protocol

1. Preparation and collection of aged worms for cell isolation

1. Collect worms and prepare buffers for cell isolation.
   1. Collect worms in a 15 mL conical tube. Wash worms from plate with 1.5 mL of M9 buffer (1 mM MgSO₄, 85 mM NaCl, 42 mM Na₂HPO₄·7H₂O, 22 mM KH₂PO₄, pH 7.0) and centrifuge for 5 min at 1,600 x g. Discard supernatant and wash worms with 1 mL of M9 buffer. Repeat centrifugation and wash for a total of five times to remove as much Escherichia coli contamination as possible.
      ​NOTE: Adding....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
6-well plate	Fisher Scientific	12-556-004
Agar, Molecular Biology Grade	VWR	A0930
CaCl2	Sigma	C5670
Contess Automated Cell Counter	Invitrogen	Z359629
DTT	USBiological	D8070
FBS	Omega Scientific	FB-02
HEPES	Sigma	H3375
KCl	Amresco	O395
KH2PO4	USBiological	P5110
Kimwipes	Kimberly-Clark Professionals	7552
Leibovitz's L-15 Medium	Gibco	21083027
MgCl2	Sigma	M8266
MgSO4	USBiological	M2090
Na2HPO4·7H2O	USBiological	S5199
NaCl	VWR	X190
NaOCl (Bleach)	Clorox
NaOH	Amresco	O583
Penicillin-Streptomicen	Fisher Scientific	15140122
Peptone Y	USBiological	P3306
Pronase E	Sigma	7433	protease mixture from Streptomyces griseus
SDS	Amresco	O227
SMT1-FLQC fluorescence stereomicroscope	Tritech Research
Sucrose	USBiological	S8010