Protocol for the assessment of mTOR activity in mouse primary hepatocytes

. 2021 Oct 29;2(4):100918. doi: 10.1016/j.xpro.2021.100918

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb (dilution 1:500)	Cell Signaling Technology	Cat#9234; RRID: AB_2269803
P70 S6 Kinase (49D7) Rabbit mAb (dilution 1:500)	Cell Signaling Technology	Cat#2708; RRID: AB_390722
Phospho-S6 Ribosomal Protein (Ser235/236) Antibody (dilution 1:1000)	Cell Signaling Technology	Cat#2211; RRID: AB_331679
S6 Ribosomal protein (5G10) Rabbit mAb (dilution 1:1000)	Cell Signaling Technology	Cat#2217; RRID: AB_331355
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (dilution 1:500)	Cell Signaling Technology	Cat#2855
4E-BP1 (53H11) Rabbit mAb (dilution 1:1000)	Cell Signaling Technology	Cat#9644; RRID: AB_2097841
Phospho-Akt (Ser473) (D9E) XP Rabbit mAb (dilution 1:1000)	Cell Signaling Technology	Cat#4060; RRID: AB_2315049
Akt (pan) (C67E7) Rabbit mAb (dilution 1:1000)	Cell Signaling Technology	Cat#4691; RRID: AB_915783
Vinculin (dilution 1:5000)	Sigma-Aldrich	Cat#V9131; RRID: AB_477629

Chemicals, peptides, and recombinant proteins

Collagen, Type I solution from rat tail	Sigma-Aldrich	Cat#C3867
PBS – Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	Cat#D8537
Penicillin-Streptomycin (5,000 U/mL)	Thermo Fisher	Cat#15070063
Gentamycin solution 50 mg/mL	Sigma-Aldrich	Cat#G1397
HBSS (1×), no calcium, no magnesium, no phenol red	Thermo Fisher Scientific	Cat#14175053
HEPES Buffer 1M	Lonza	Cat#BE17-737E
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid	Sigma-Aldrich	Cat#E3889
William’s E Medium, no glutamine	Thermo Fisher Scientific	Cat#22551022
Collagenase, Type I	Worthington-biochem	Cat#LS004196
Percoll	Sigma-Aldrich	Cat#GE17-0891-01
HBSS (10×), no calcium, no magnesium, no phenol red	Thermo Fisher Scientific	Cat#14185052
DMEM, high glucose	Thermo Fisher Scientific	Cat#41965039
Ham’s F-12 Nutrient Mix	Thermo Fisher Scientific	Cat#21765029
Bovine Serum Albumin essentially fatty acid free	Sigma-Aldrich	Cat#A6003
Sodium bicarbonate (NaHCO₃)	Merck	Cat#1063291000
Sodium pyruvate solution 100 mM	Sigma-Aldrich	Cat#S8636
L-Glutamine solution 200 mM	Sigma-Aldrich	Cat#G7513
Fetal Bovine Serum (FBS)	Hyclone	Cat#SV30160.03
Dulbecco’s MEM (DMEM) F-12 without Amino acids, Glucose, L-Glutamine, Sodium Bicarbonate, HEPES, Sodium	USBiological Life Sciences	Cat#D9807-10
D-(+)-Glucose solution	Sigma-Aldrich	Cat#G8769
C (L-Cystine-2HCl)	Sigma-Aldrich	Cat#C6727
F (L-Phenylalanine)	Sigma-Aldrich	Cat#P5482
H (L-Histidine)	Sigma-Aldrich	Cat#H5659
I (L-Isoleucine)	Sigma-Aldrich	Cat#I7403
K (L-Lysine-HCl)	Sigma-Aldrich	Cat#L8662
L (L-Leucine)	Sigma-Aldrich	Cat#L8912
M (L-Methionine)	Sigma-Aldrich	Cat#M5308
Q (L-Glutamine)	Sigma-Aldrich	Cat#G3126
R (L-Arginine)	Sigma-Aldrich	Cat#A8094
T (L-Threonine)	Sigma-Aldrich	Cat#T8441
V (L-Valine)	Sigma-Aldrich	Cat#V0513
W (L-Tryptophan)	Sigma-Aldrich	Cat#T8941
Y (L-Tyrosine-2Na-2H₂O)	Sigma-Aldrich	Cat#T8566
D (L-Aspartic Acid)	Sigma-Aldrich	Cat#A8949
E (L-Glutamic Acid)	Sigma-Aldrich	Cat#G1251
G (Glycine)	Sigma-Aldrich	Cat#G7126
N (L-Asparagine)	Sigma-Aldrich	Cat#A0884
P (L-Proline)	Sigma-Aldrich	Cat#P0380
S (L-Serine)	Sigma-Aldrich	Cat#S4500
HEPES	Sigma-Aldrich	Cat#H3375
Sodium chloride	Sigma-Aldrich	Cat#S9625
EDTA 0.5 M, pH 8	Thermo Fisher Scientific	Cat#15575020
Sodium orthovanadate (NaVO₄)	Sigma-Aldrich	Cat#S6508
Sodium Fluoride (NaF)	Sigma-Aldrich	Cat#S7920
Sodium Pyrophosphate tetra-basic decahydrate	Sigma-Aldrich	Cat#S6422
β-Glycerophosphate disodium salt hydrate	Sigma-Aldrich	Cat#G9422
Triton X-100	Sigma-Aldrich	Cat#T8787
Sodium deoxycholate	Sigma-Aldrich	Cat#D6750
Complete Mini Protease Inhibitor Cocktail	Roche	Cat #11836153001
Sodium dodecyl sulfate (SDS)	Merck	Cat#151-21-3
70% Ethanol	N/A	N/A
Ketamine	Imalgene 1000	N/A
Xylazine	Rompur	N/A
Physiological saline solution	B Braun	Cat #607189.2
Trypan Blue solution	Sigma-Aldrich	Cat#T8154
Insulin human	Sigma-Aldrich	Cat#I2643
SnakeSkin Dialysis Tubing, 3.5K MWCO, 35 mm dry I.D., 35 feet	Thermo Fisher Scientific	Cat #88244

Critical commercial assays

Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	Cat#23225
Primary mouse hepatocytes	This paper	N/A

Experimental models: Organisms/strains

C57BL/6J (7–10 weeks)	Jackson Laboratory	Cat#JAX:000664; RRID: IMSR_JAX:000664

Software and algorithms

ImageJ software	ImageJ	https://imagej.nih.gov/ij/

Other

6-well Clear Multiwell Plate	Life Sciences	Cat#353046
500 mL Vacuum Filter/Storage Bottle System, 0.22 μm Pore 33.2 cm² PES Membrane, Sterile, 12/Case	Life Sciences	Cat#431097
Corning Polypropylene Centrifuge Tubes, Sterile (50 mL)	Fisher Scientific	Cat#10509891
Masterflex C/L Analog Variable-Speed Pump with Dual-Channel Pump Head for Microbore Tubing Pump, 10–60 rpm; 12 VDC	Cole-Parmer	Cat#EW-77120-62
Water bath Thermo Haake	Thermo Fisher Scientific	Cat#003–2859
Sterile needles 23G ×1” – Nr. 16 0.6 mm × 25 mm	BD Microlance 3	Cat#300800
Sterile surgical non-absorbable sutures 2/0 USP 90 cm	LorcaMarín	Cat#55184-50U
Intravascular Catheter BD Vialon 22GA 0.9 × 25 mm	Clinimark SL	Cat#381223
ROCKER 3D digital (Shaker)	IKA	Cat#0004001000
Fine Scissors – Sharp-Blunt	Fine Science Tools	Cat#14028-10
Dumont #5 – Fine Forceps	Fine Science Tools	Cat#11254-20
Corning 100 μm Cell Strainer, Yellow, Sterile, Individually Packaged, 50/Case	Life Sciences	Cat#431752
TC20 Automated cell counter	Bio-Rad	Cat#145-0101
Cell Lifter with J-Hook Blade	VWR	Cat#76036-004
Eppendorf 5810 Centrifuge	Merck	Cat#EP5810000320
Eppendorf 5424R Refrigerated Centrifuge	Marshall Scientific	Cat#EPP-5424R
ELMI RM-2L Intelli-mixer Large	ELMI	Cat#ELMI RM-2L
Parafilm	Merck	Cat#P7793