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. 2021 Aug 18;7:76. doi: 10.1038/s41531-021-00210-w

Table 1.

Biochemical assays.

Dissociation media composition
Chemical name	Concentration	Vendor	Catalog number
NaCl	116 mM	Sigma-Aldrich	S7653
NaHCO₃	26 mM	Sigma-Aldrich	S5761
NaH₂PO₄	2 mM	Sigma-Aldrich	S9638
d-glucose	25 mM	Sigma-Aldrich	G8769
MgSO₄	1 mM	Sigma-Aldrich	M7506
Cysteine	1.3 mM	Sigma-Aldrich	C7352
Papain	400 U/ml	Worthington Biochemical Corporation	LS003127
Kynurenic acid	0.5 mM	Sigma-Aldrich	K3375

Glia media composition
Chemical name	Concentration	Vendor	Catalog number
DMEM	51.45%	Thermo Fisher Scientific	11330032
Fetal bovine serum	39.6%	Gemini	100–106
Penicillin/streptomycin	1%	Thermo Fisher Scientific	15-140-122
Glutamax 100X	1%	Thermo Fisher Scientific	35050061
Insulin (25 mg/ml stock)	0.08%	Sigma-Aldrich	I5500

DIV0 neuronal media composition
Chemical name	Concentration	Vendor	Catalog number
Neurobasal-A	96.9%	Thermo Fisher Scientific	10888022
B27 Plus	1.9%	Thermo Fisher Scientific	A3582801
GDNF	0.15%	Sigma-Aldrich	SRP3200
Glutamax 100X	1%	Thermo Fisher Scientific	35050061
Kynurenic acid	0.08%	Sigma-Aldrich	K3375

Maintenance of neuronal media composition
Chemical name	Concentration	Vendor	Catalog number
Neurobasal-A	97.1%	Thermo Fisher Scientific	10888022
B27 Plus	1.9%	Thermo Fisher Scientific	A3582801
Glutamax 100X	0.97%	Thermo Fisher Scientific	35050061

Immunocytochemistry
General reagents
Chemicals	Concentration	Vendor	Catalog number
PBS	1X	Prepared as needed on-site	N/A
Triton X-100	0.50%	Thermo Fisher Scientific	bp151-100
NGS	10%	Lampire Biological Products	7332500
Paraformaldehyde	4%	Electron Microscopy Sciences	157-4-100

Primary antibodies
Primary antibody–antigen	Host species/isotype	Concentration	Vendor	Catalog number	Use
Tyrosine hydroxylase	Rabbit/polyclonal	1:500	EMD Millipore	AB152	ICC/WB
Alpha-synuclein (syn211)	Mouse/IgG1	1:500	Abcam	AB80627	ICC
DAT	Rat	1:500	Millipore Sigma	AB5802
Calbindin	Rabbit/polyclonal	1:500	EMD Millipore	ABN2192
GFP	Rabbit/IgG	1:500	Thermo Fischer Scientific	A-11122	ICC
Human α-syn (C-terminal regions, 130–140) (94-3A10)	Mouse/monoclonal IgG1	1:500	Gifted from Dr. Giasson	N/A	WB
Actin C4	Mouse/monoclonal	1:500	EMD Millipore	MAB1501	WB
D2	Mouse	1:500	Neuromab	75–230	WB/ICC
D2	Rabbit	1:500	Millipore	AB5084P	WB

Secondary antibodies
Conjugate	Host species/target species	Concentration	Vendor	Catalog number	Use
Alexa Fluor 568	Goat/mouse	1:500	Life Technologies	A-21124	ICC
Alexa Fluor 647	Goat/mouse	1:500	Life Technologies	A21242	ICC
Alexa Fluor 488	Goat/rabbit	1:500	Life Technologies	A-11006	ICC
HRP	Goat/mouse	1:2000	Jackson Immuno Research Labs	AB-10015289	WB
HRP	Goat/rabbit	1:2000	Jackson Immuno Research Labs	AB_2307391	WB
IRDye® 800CW	Goat/mouse	1:15000	LI-COR	926-32210	WB

Biotinylation, cell lysis, and ELISA
ELISA coating buffer, pH 9.6
Chemical name	Concentration	Vendor	Catalog number
Na₂CO₃	28.3 mM	Sigma-Aldrich	D6546
NaHCO₃	71.42 mM	Sigma-Aldrich	S5761

BufferD lysis buffer, pH 7.6
Chemical name	Concentration	Vendor	Catalog number
Glycerol	10% (v/v)	Sigma-Aldrich	G5516
NaCl	125 mM	Sigma-Aldrich	S7653
EDTA	1 mM	Sigma-Aldrich	E9884
EGTA	1 mM	Sigma-Aldrich	3777

Biotinylation (buffer pH 7.8)
Chemical name	Concentration	Vendor	Catalog number
NaCl	150 mM	Sigma-Aldrich	S7653
CaCl₂	2 mM	Sigma-Aldrich	449709
Triethanolamine	10 mM	Sigma-Aldrich	90279
Sulfo-NHS-SS biotin	1.5 mg/ml	Thermo Fisher Pierce	22331
Monomeric avidin resin	N/A	Thermo Fisher Pierce	53146

Antibodies for ELISA
Specificity	Clone/species	Conjugate	Dilution	Vendor	Catalog number
TH	Monoclonal/mouse	N/A	1:1000	EnCor	MCA-4H2
TH	Polyclonal/rabbit	Biotin	1:1000	EnCor	RPCA-TH
Biotin	N/A (avidin)	HRP	1:2500	Vector Labs	A-2004

Calcium imaging ACSF composition
pH range: 7.35–7.4; osmolarity range: 305–310
Chemical name	Concentration	Vendor	Catalog number
NaCl	126 mM	Sigma-Aldrich	S7653
KCl	2.5 mM	Sigma-Aldrich	P9541
CaCl₂	2 mM	Sigma-Aldrich	223506
NaH₂PO₄	1.25 mM	Sigma-Aldrich	71505
MgSO₄	2 mM	Sigma-Aldrich	M7506
Dextrose	10 mM	Sigma-Aldrich	D9434
NaHCO₃	24 mM	Sigma-Aldrich	S5761

Pharmacological agents
Resource/reagent	Concentration	Vendor	Catalog number
Dopamine hydrochloride	1 μM	Sigma-Aldrich	H8502
Quinpirole hydrochloride	10 μM	Sigma-Aldrich	Q102

Electrophysiology ACSF composition
pH range: 7.35–7.4; osmolarity range: 305–310
Chemical name	Concentration	Vendor	Catalog number
NaCl	126 mM	Sigma-Aldrich	S7653
KCl	2.5 mM	Sigma-Aldrich	P9541
CaCl₂	2 mM	Sigma-Aldrich	223506
NaH₂PO₄	1.25 mM	Sigma-Aldrich	71505
MgSO₄	2 mM	Sigma-Aldrich	M7506
Dextrose	10 mM	Sigma-Aldrich	D9434
NaHCO₃	26 mM	Sigma-Aldrich	S5761
Potassium gluconate	120 mM	Sigma-Aldrich	P1847
MgCl₂	2 mM	Sigma-Aldrich	M8266
HEPES	10 mM	Sigma-Aldrich	H3375
EGTA	0.1 mM	Sigma-Aldrich	E3889
ATPNa₂	2 mM	Sigma-Aldrich	A2383
GTPNa	0.25 mM	Sigma-Aldrich	A2383

Reagents for ELISA
Reagent	Concentration	Supplier	Catalog number	Purpose
Fat-free milk	1% or 5%>	Carnation	N/A	WB/ELISA
TMB substrate	Stock	Thermo Fisher	34028	ELISA
H₂SO₄	2 N	Sigma	339741	ELISA
Tween-20	0.002	Thermo Fisher	MP1Tween201	TBS-T
Protease inhibitor	1×	Milipore	539191	Cell lysis
DC protein assay	N/A	Biorad	5000112	Protein assay
Immulon 4 HBX	N/A	Thermo Fisher	3855	ELISA

1× KH buffer (cell lysate)
pH: 7.4, adjusted with HCl or NaOH as needed
Chemical name	Concentration	Vendor	Catalog number
NaCl	118 mM	Sigma-Aldrich	S7653
KCl	4.7 mM	Sigma-Aldrich	P9541
CaCl₂	1.25 mM	Sigma-Aldrich	223506
KH₂PO₄	1.2 mM	Sigma-Aldrich	P5655
MgSO₄	1.2 mM	Sigma-Aldrich	M7506
d-glucose	11 mM	Sigma-Aldrich	G8769
l-Ascorbic acid	0.5 mg/mL	Sigma-Aldrich	A5960
NaHCO₃	25 mM	Sigma-Aldrich	S5761

HEPES ACSF
pH: 7.4, adjusted with HCl or NaOH as needed
Chemical name	Concentration	Vendor	Catalog number
NaCl	92 mM	Sigma-Aldrich	S7653
KCl	2.5 mM	Sigma-Aldrich	P9541
CaCl₂	0.5 mM	Sigma-Aldrich	223506
NaH₂PO₄	1.2 mM	Sigma-Aldrich	P5655
NaHCO₃	30 mM	Sigma-Aldrich	S5761
MgSO₄	10 mM	Sigma-Aldrich	M7506
d-glucose	25 mM	Sigma-Aldrich	G8769
HEPES	20 mM	Sigma-Aldrich	H3375
Na-l-ascorbate	5 mM	Sigma-Aldrich	A4034
Na-pyruvate	3 mM	Sigma-Aldrich	P5280
Thiourea	2 mM	Sigma-Aldrich	T7875

PBS phosphate-buffered saline, DAT dopamine transporter, GFP green fluorescent protein, NGS normal goat serum, N/A not available.