Establishing a Müller Glial Cell Culture Obtained from Mouse Retinas

Samenvatting

Source: Kang, S., et al. Primary Cell Cultures to Study the Regeneration Potential of Murine Müller Glia after MicroRNA Treatment. J. Vis. Exp. (2022).

This video demonstrates the culturing of Müller glial (MG) cells isolated from mouse retinas. Retinas are dissociated into a single-cell suspension using digestive enzymes. Cells are then grown in a medium with a growth factor that promotes MG cell proliferation and neuronal cell mortality. Dead neuronal cells are removed while passaging, and MG cells are grown for further analysis.

Protocol

Disclaimer: All procedures involving sample collection have been performed in accordance with the institute’s IRB guidelines.

1. Retina dissociation

NOTE: All following steps (until cell harvest) need to be carried out in an A2 or B2 biosafety cabinet (BSC).

1. Prepare the Papain/DNase I dissociation mixture as follows.
   1. For six retinas, add 75 µL of DNase I into the tube containing 750 µL of Papain and mix carefully (dissociation mixture).
   2. For individual sample preparation required for this protocol, split the tota....

Resultaten

Figure 1: Retinal dissociation and genotyping. (A) Eye cup with removed cornea, lens, iris, and vitreous. (B) Isolated retinas; retinal pigment epithelial (RPE) cells are removed after a thorough wash. (C) 12-well plate with dissociated retinas (two re.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Animals
Ascl1-CreERT mouse Ascl1tm1.1(Cre/ERT2)Jejo/J	Jax laboratories	#012882	Ascl1-CreERT mice were crossed with tdTomato mice
tdTomato-STOPfl/fl mouse B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J	Jax laboratories	#007914	Genotyping is requried to identify Ascl1CreER positive mice
Reagents
Poly-L-ornithine hydrobromide	Sigma-Aldrich	P4538-50MG	reconstituted in steriled water, frozen aliquots
N-2 Supplement	Fisher Scientific	17-502-048	frozen aliquots
Neurobasal Medium	Fisher Scientific	21-103-049	used for growth medium in section 1.1, store at 4 °C
Papain Dissociation System	Worthington Biochemical	LK003153	reconstituted in Earle's Balanced Salt Solution, frozen aliquots
Penicillin Streptomycin	Fisher Scientific	15-140-122	frozen aliquots
L-Glutamine	Fisher Scientific	25-030-081	frozen aliquots
HBSS	Fisher Scientific	14-025-134	store at 4 °C
Fetal Bovine Serum (FBS)	Fisher Scientific	MT35010CV	frozen aliquots
plasticware/supplies
0.6 mL microcentrifuge tube	Fisher Scientific	50-408-120
1.5 mL microcentrifuge tube	Fisher Scientific	50-408-129
10 µL TIP sterile filter Pipette Tips	Fisher Scientific	02-707-439
100 µL TIP sterile filter Pipette Tips	Fisher Scientific	02-707-431
1000 µL TIP sterile filter Pipette Tips	Fisher Scientific	02-707-404
2.0 mL microcentrifuge tube	Fisher Scientific	50-408-138
20 µL TIP sterile filter Pipette Tips	Fisher Scientific	02-707-432
Adjustable-Volume Pipettes (2.5, 10, 20, 100, 200, & 1000 µL)	Eppendorf	2231300008
Disposable Transfer Pipets	Fisher Scientific	13-669-12
Multiwell Flat-Bottom Plates with Lids, No. of Wells=12	Fisher Scientific	08-772-29
Multiwell Flat-Bottom Plates with Lids, No. of Wells=24	Fisher Scientific	08-772-1
PIPET sterile filter 10ML Disposable Serological Pipets	Fisher Scientific	13-676-10J
PIPET sterile filter 50ML Disposable Serological Pipets	Fisher Scientific	13-676-10Q
PIPET sterile filter 5ML Disposable Serological Pipets	Fisher Scientific	13-676-10H
Powder-Free Nitrile Exam Gloves	Fisher Scientific	19-130-1597B
Round coverslips (12 mm diameter, 0.17 - 0.25 mm thickness)	Fisher Scientific	22293232
Vacuum Filter, Pore Size=0.22 µm	Fisher Scientific	09-761-106
equipment
1300 B2 Biosafety cabinet	Thermo Scientific	1310
All-in-one Fluorescence Microscope Keyence BZ-X 810	Keyence	9011800000
Binocular Zoom Stereo Microscope	Vision Scientific	VS-1EZ-IFR07
Disposable Petri Dishes (100 mm diameter)	VWR	25384-088
Dumont #5 Forceps - Biologie/Titanium	Fine Science Tools	11252-40
Dumont #55 Forceps - Biologie/Inox	Fine Science Tools	11255-20
Dumont #7 curved Forceps - Biologie/Titanium	Fine Science Tools	11272-40
Eppendorf Centrifuge 5430 R	Eppendorf	2231000508
Water Jacketed CO2 Incubator	VWR	10810-744