Isolation and Culture of Rat Tanycytes

Tanycytes were isolated from the median eminence of the hypothalamus of 10-d-old rats and cultured as described previously ^{54 (link),84 (link)}. Briefly, after decapitation and removal of the brain, median eminences were dissected and crushed on 80μM nylon mesh (Sefar America Inc., Kansas City, MO). Dissociated cells were cultured in DMEM/F12 (Invitrogen, Cergy Pontoise, France) supplemented with 10% (v/v) donor calf serum (Invitrogen) under humid atmosphere of 5 % CO2-95 % air at 37 °C. Culture medium was changed after 3-4 days of culture and subsequently every 2 days. Upon reaching confluence, the tanycytes were isolated from contaminating cells by overnight shaking at 250 rpm at 37 °C and either replated in 6 cm dishes for Western blot experiments or seeded in culture plates on poly-L-lysine-coated glass coverslips for studying leptin traficking. Two days before treatment, the medium was replaced by a tanycyte defined medium (TDM) consisting of DMEM/F12 (devoid of phenol red; Invitrogen) supplemented with insulin (5μg/ml) (Sigma, Saint Quentin Fallavier, France) and putrescin (100μM) (Sigma).

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Duquenne M., Folgueira C., Bourouh C., Millet M., Silva A., Clasadonte J., Imbernon M., Fernandois D., Martinez-Corral I., Kusumakshi S., Caron E., Rasika S., Deliglia E., Jouy N., Oishi A., Mazzone M., Trinquet E., Tavernier J., Kim Y.B., Ory S., Jockers R., Schwaninger M., Boehm U., Nogueiras R., Annicotte J.S., Gasman S., Dam J, & Prévot V. (2021). Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function. Nature metabolism, 3(8), 1071-1090.

Publication 2021

DMEM/F12 donor calf serum insulin putrescin

Atmosphere Brain Cells Decapitation Dishes Donor Insulin Leptin Lysine Median eminences Nylon Poly Putrescin Rats Serum Tanycyte Western blot

Corresponding Organization :

Other organizations : Université de Lille, Centre de Recherche Jean Pierre Aubert, Instituto de Investigación Sanitaria de Santiago, Universidade de Santiago de Compostela, Institut des Neurosciences Cellulaires et Intégratives, Institut Cochin, Saarland University, KU Leuven, VIB-KU Leuven Center for Cancer Biology, Cisbio (France), VIB-UGent Center for Medical Biotechnology, Beth Israel Deaconess Medical Center, Institut Pasteur de Lille

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Variable analysis

independent variables

Isolation of tanycytes from the median eminence of the hypothalamus of 10-d-old rats
Culture conditions (DMEM/F12 supplemented with 10% donor calf serum, cultured under 5% CO2-95% air at 37°C)
Overnight shaking at 250 rpm at 37°C to isolate tanycytes from contaminating cells
Replacement of medium with tanycyte defined medium (TDM) consisting of DMEM/F12, insulin (5μg/ml), and putrescin (100μM) two days before treatment

dependent variables

Leptin trafficking in tanycytes

control variables

Tissue source (median eminence of the hypothalamus of 10-d-old rats)
Culture medium (DMEM/F12 supplemented with 10% donor calf serum)
Culture conditions (5% CO2-95% air at 37°C)
Isolation of tanycytes from contaminating cells by overnight shaking at 250 rpm at 37°C

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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