Integrated functional neuronal network analysis of 3D silk-collagen scaffold-based mouse cortical culture

. 2021 Jan 25;2(1):100292. doi: 10.1016/j.xpro.2020.100292

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains

AAV-hSyn1-GCaMP6f-P2A-nls-dTomato	Addgene	51085-AAV1 https://www.addgene.org/51085/
pENN.AAV.hSyn.TurboRFP.WPRE.RBG	Addgene	105552-AAV1 https://www.addgene.org/105552/

Chemicals, peptides, and recombinant proteins

Sodium carbonate (Na₂CO₃)	MilliporeSigma	57795
Lithium bromide (LiBr)	MilliporeSigma	213225
Sodium chloride (NaCl)	MilliporeSigma	S7653
Poly-D-lysine (PDL)	MilliporeSigma	P6407
Laminin (0.5 mg/mL)	MilliporeSigma	MFCD00081739
Collagen type I solution	Corning	354236
Sodium hydroxide (NaOH)	MilliporeSigma	S2770
Calcein-AM	Thermo Fisher	C3100MP
Picrotoxin	Abcam	ab120315
Bicuculline	Abcam	ab120107
2,3-Dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX)	Tocris	1044
(2R)-Amino-5-phosphonopentanoate (AP5)	MilliporeSigma	A8054
Tetrodotoxin citrate	Abcam	ab120055
β-Mercaptoethanol	MilliporeSigma	M3148
Dimethyl sulfoxide	MilliporeSigma	D2650
0.25% Trypsin-EDTA	Thermo Fisher	25200
DNase	MilliporeSigma	10104159001
Hank's balanced salt solution (HBSS)	Thermo Fisher	14025
Phosphate-buffered saline (PBS), sterile	Thermo Fisher	10010
Neurobasal medium minus phenol red	Thermo Fisher	12348
B27 supplement	Thermo Fisher	15240
Antibiotic-antimycotic	Thermo Fisher	15240
GlutaMax	Thermo Fisher	35050
Fetal bovine serum (FBS)	Thermo Fisher	10437
10× PBS	Fisher Scientific	BP399-4

Oligonucleotides

Gabra1 (Taqman Assay)	Thermo Fisher	Mm00439046_m1
Gad2 (Taqman Assay)	Thermo Fisher	Mm00484623_m1
Gria1 (Taqman Assay)	Thermo Fisher	Mm00433753_m1
Grin1 (Taqman Assay)	Thermo Fisher	Mm00433790_m1
Pax6 (Taqman Assay)	Thermo Fisher	Mm00443081_m1
Rn18s (Taqman Assay)	Thermo Fisher	Mm03928990_g1
Shank3 (Taqman Assay)	Thermo Fisher	Mm00498775_m1
Slc17a7 (Taqman Assay)	Thermo Fisher	Mm00812886_m1
Slc1a3 (Taqman Assay)	Thermo Fisher	Mm00600697_m1
Syn1 (Taqman Assay)	Thermo Fisher	Mm00449772_m1
Dcx (Taqman Assay)	Thermo Fisher	Mm00438400_m1

Software and algorithms

FluoroSNNAP	https://www.seas.upenn.edu/∼molneuro/software.html	n/a
Photoshop	Adobe	n/a
Illustrator	Adobe	n/a
NIS-Elements AR	Nikon	n/a
MATLAB	MathWorks	n/a
MATLAB Bioinformatics Toolbox Add-on	https://www.mathworks.com/products/bioinfo.html	n/a

Deposited data

RunNetworkAnalysis MATLAB Code	https://github.com/mattiabonzanni/Integrated-Functional-Neuronal-Network-Analysis-of-3D-Silk-Collagen-Scaffold-based-Cortical-Culture	n/a

Other

Weigh dishes (small, medium, large)	Fisher Scientific	02-202-100, 02-202-101, 02-202-102
Tissue culture dishes (35-mm, 60-mm, 100-mm)	Corning	430165, 430166, 430167
Syringe filters (0.22-μm, sterile)	MilliporeSigma	SLGV033RB
Syringes (5 mL)	Fisher Scientific	14-817-53
Cell strainers, 100-μm	Corning	431752
Bombyx mori cocoons	Tajima Shoji Co., Ltd, Japan	n/a
Dialysis tubing, molecular weight cutoff 3,500 Da	Fisher Scientific	21-152-9
Dialysis clips	Thermo Fisher	68011
Stainless steel spatula	Fisher Scientific	21-401-10
Biopsy punch, 3-mm diameter	Integra	12-460-406
Razor blades	Fisher Scientific	12-640
Scalpel blades	Fisher Scientific	NC9855170
Surgical scissors	Fine Science Tools	14060-09
Tissue forceps	Fine Science Tools	11052-10
Tissue culture plates (12-, 24-, 48-, 96-wells)	Fisher Scientific	08-772-29, 08-772-1, 08-772-1C, 08-7722c
Conical tubes (15 mL, 50 mL)	Fisher Scientific	14-959-53a, 14-432-22
Tissue culture plates, glass-bottom, 24-well	Cellvis	P24-1.5P
Nuclease-free 1.5 mL microcentrifuge tubes	Fisher Scientific	05-408-129
Homogenizer pestle	Fisher Scientific	12-141-364
Motorized tissue grinder	Fisher Scientific	12-1413-61
QIAshredder column	Qiagen	79656
Nuclease-free 0.2 mL PCR tubes or strips	Costar	07-200-681
Dumont #5 forceps	Fine Science Tools	11251-35 or11252-50
Angled forceps	Fine Science Tools	11251-35
3 L stainless steel beakers	Polar Ware	3W
4 L plastic beakers	Thermo Fisher	12014000
Float Buoys	Thermo Fisher	66432
Conductivity meter	Fisher Scientific	15-078-201
Stainless steel sieves (425 μm, 500 μm, lid/receiver)	Fisher Scientific	04-884-1AP, 04-884-1AN, 04-888A
Sterile, nuclease-free water	Thermo Fisher	10977015
Glass beaker (small)	Fisher Scientific	FB100100
Glass graduated cylinder	Fisher Scientific	08-550D
Dissection microscope	Nikon or equivalent	SMZ-754T or equivalent
Epifluorescence microscope equipped with a 4× objective, green fluorescence filter cube, and time-lapse acquisition function	Nikon or equivalent	Nikon Eclipse Ti-2 with Objective: CFI Plan Fluor DL 4× na 0.13 wd 16.5 mm Objective, filter #49002, and NIS-Elements AR software, or equivalent
Microscope stage-top incubator with temperature and CO₂ controls	Bioscience Tools or equivalent	TC-MWP or equivalent
Confocal microscope with excitation laser and detector for red fluorescence	Leica or equivalent	SP8 or equivalent
NanoDrop UV-vis spectrophotometer	Thermo Fisher	2000 or equivalent
Thermocycler	Bio-Rad or equivalent	T100 or equivalent
qRT-PCR machine	Thermo Fisher	QuantStudio 5 or equivalent
3D Printer	Formlabs or equivalent	Form2 or equivalent *Alternative:* Order 3D printed parts from ShapeWays.com

Critical commercial assays

Qiagen RNeasy Mini Kit	Qiagen	79656
iScript gDNA Clear cDNA Synthesis Kit	Bio-Rad	1725035
Taqman Gene Expression Master Mix	Thermo Fisher	4369016

Experimental models: organisms/strains

Timed-pregnant (E16.5) C57BL/6 mouse	Charles River Laboratories	027