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Sterilization pouches (VWR, catalog number: 58753-194)
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Transfer pipette (VWR, catalog number: 414004-002)
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Glass Pasteur pipette (VWR, catalog number: 14673-043)
Plug the wide end with of the glass pipette with cotton. Polish the capillary end using a Bunsen burner by holding the tip in the flame and rotating it rapidly. Pause every few seconds and check the size of the opening to ensure that the edge is smooth and round and the opening is not too narrow. A narrow opening will disrupt cells during the trituration. Autoclave the fire polished Pasteur pipette to sterilize before use.
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35 mm Petri dishes (VWR, Greiner Bio-One, catalog number: 82050-540)
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100 mm Petri dishes (VWR, catalog number: 25384-342)
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150 mm Petri dishes (Midland Scientific Inc., Kord Valmark, catalog number: VM 2902)
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Microfluidic devices (Xona microfluidics, catalog number: XC450)
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Glass bottom dishes (MatTek, catalog number: P35G-1.5-14-C)
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Closures for dialysis membrane tubing (VWR, Spectrum Laboratories [132735], catalog number: 25224-075)
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Glass vial (Qorpak, catalog number: GLC-00987)
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Dialysis membrane tubing, MWCO 12000-14000 (Fisher Scientific, SpectrumTM S432697, catalog number: 08-667A)
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Syringe filter 0.22 μm (VWR, catalog number: 28145-477)
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Conical tubes (VWR, Corning, catalog number: 21008-656)
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Eppendorf tubes (FisherbrandTM, catalog number: 05408129)
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E14.5 pregnant Sprague Dawley rat
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Immersion oil for microscopy (Cargille, catalog number: 16484)
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Papain (Worthington Biochemical, catalog number: LS003126)
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L-cysteine (Sigma-Aldrich, catalog number: C7352)
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Ovomucoid protease inhibitor (Worthington, catalog number: LK003182). Reconstitute in 32 ml of HBSS and store at 4 °C
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Collagenase A (Roche, catalog number: 10103578001)
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DNase I (Sigma-Aldrich, Catalog number: D5025-15KU)
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Trypan blue (Sigma-Aldrich, catalog number: T8154)
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Hanks’ balanced salt solution without calcium and magnesium, HBSS (CellGro-Corning, catalog number: 21-022-CV)
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Poly-D-lysine hydrobromide, PDL (Sigma-Aldrich, catalog number: P0899). Reconstitute in HBSS at a concentration of 0.5 mg/ml, filter sterilize, make 5 ml aliquots in sterile conical tubes and store at -20 °C
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Laminin (Corning, catalog number: 354232)
Thaw slowly on ice, make aliquots of different volumes (10 to 50 μl) in sterile tubes, and store at -20 °C. Laminin will form a gel following rapid changes in temperature. For coating, combine multiple aliquots of different volumes to arrive at the final required volume. Do not re-freeze after thawing.
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Alexa FluorTM 647 NHS Ester (Thermo Fisher Scientific, catalog number: A37573). Reconstitute at 10 mM concentration in DMSO using sterile conditions and make 2 μl aliquots. Store at -20 °C and use within 6 months. The dye solution becomes hydrolyzed over time, resulting in poor labeling efficiency. Do not refreeze after thawing.
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Sodium bicarbonate, NaHCO3 (Fisher chemicalTM, catalog number: S233-500)
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NeurobasalTM medium (Gibco, catalog number: 21103049)
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UltraCULTURETM serum-free (Lonza, catalog number: BE12-725F)
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GlutaMAXTM supplement (Gibco, catalog number: 35050079)
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Penicillin-streptomycin (Gibco, catalog number: 15070-063)
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HyCloneTM fetal bovine serum (GE Healthcare/Cytiva, catalog number: SH30088.03). Make 50 ml aliquots in sterile conical tubes and store at -20 °C. Store thawed aliquots at 4 °C up to 6 months
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N-2 supplement (Gibco, catalog number: 17502048). Thaw on ice, make 500 μl aliquots in sterile tubes, and store at -20 °C
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B-27TM supplement (Gibco, catalog number: 17504044). Thaw on ice, make 1 ml aliquots in sterile tubes, and store at -20 °C
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Nerve growth factor 2.5S subunit, NGF (Gibco, catalog number: 13257019). Thaw on ice, make 15 μl aliquots in sterile tubes, and store at -20 °C. Supplied at 100 µg/ml
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Hibernate-E medium (Gibco, catalog number: A1247601)
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Cytosine β-D-arabinofuranoside, AraC (Sigma-Aldrich, catalog number: C1768). Reconstitute in water at a concentration of 10 mM, filter sterilize, make 100 μl aliquots in sterile tubes, and store at -20 °C. To limit repeated freeze-thaws, each aliquot can be re-aliquoted in 10 μl upon thawing and stored at -20 °C
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NaCl (Fisher ChemicalTM, catalog number: S671-3)
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KCl (Fisher ChemicalTM, catalog number: P217-500)
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Na2HPO4 (Sigma-Aldrich, catalog number: S-7907)
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KH2PO4 (Fisher ChemicalTM, catalog number: P285-500)
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Collagenase-DNase I mixture (see Recipes)
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Base medium (see Recipes)
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Complete medium (see Recipes)
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Low NGF medium (see Recipes)
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Papain solution (see Recipes)
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10x PBS (see Recipes)
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10x sodium bicarbonate buffer (see Recipes)