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, , 2018.
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, organisation de la journée d'accueil des étudiants du Master Neurosciences de Strasbourg, 2016.
, et 2017 : animation d'ateliers scientifiques durant la Fête de la Science, 2016.
, , 2017.
, 2018 : participation à l'entraînement des étudiants du Master 2 Neurosciences de Strasbourg pour la préparation de leur soutenance ?, 2017.
, conception et animation des ateliers « Les Mystères du Cerveau », destinés à des collégiens (journée « Kids University, 2016.
, et octobre 2017 : animation de conférences sur le quotidien d'un doctorant, pour des lycéens de filière scientifique ? 2018 : participation à la demi-finale régionale du concours « Ma thèse en 180 secondes, 2016.
, The Coffin-Lowry syndrome-associated protein RSK2 regulates neuritis outgrowth through phosphorylation of phospholipase D1 (PLD1) and synthesis of phosphatidic acid, Autres activités de médiation scientifique Décembre 2018 : participation à « DECLICS » (Speed meeting scientifique avec des lycéens) Annexes References Ammar MR, vol.33, pp.19470-19479, 2013.
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, Adjustable volume pipettes with sterile tips
,
, Sterile surgical material required: forceps, scissors, and scalpel blades
, For the physical separation of cells, 250 mL centrifuge tubes are cut at the bottom; plugs are cut and used to adjust 217 µm mesh sieve filters (SAATI
, µm mesh filter (Falcon)
, Large glass tray (30 cm by 20 cm) and glass beakers 250-mL
, A centrifuge capable of reaching 20,000 g for Percoll gradient
, 96-well culture plates (Thermo Fisher Scientific, France) for cell culture
, 96-well black-plates (Thermo Scientific) for the secretion assay
, Two Eppendorf research plus adjustable volume multichannel pipettes and 5-10 multichannel disposable solutions basins
, Fluorimeter allowing excitation at 430 nm and emission at 520 nm of 96-well plates, We use a LB940 Mitras
, This solution is filtered through 0.22-µm syringe filters. For each culture, 50 mL of concentrated Krebs solution is, Ca 2+ -and Mg 2+ -free concentrated Krebs solution: 1.54 M NaCl, 56 mM KCl, 36 mM NaHCO 3 , 56 mM glucose, and 50 mM HEPES pH 7.4, p.450
, Trypan blue (Sigma): 0.4% in Krebs balanced salt solution
, Cytosine arabinoside (Sigma): 30 mg in 100 mL Dulbecco's modified Eagle's medium (DMEM, Sigma) and filter sterilized
, Fluorodeoxyuridine (Sigma): 24.6 mg in 10 mL Krebs balanced salt solution and filter sterilized
, Depending on plating needs (see bellow), this medium is further completed with 1% cytosine arabinoside and 0.1% fluorodeoxyuridine solutions and 10% fetal bovine serum previously decomplemented 30 min at 56°C (Gibco, We use standard DMEM supplemented with glutamine (Sigma) to prepare chromaffin cells
, Collagenase solution is prepared with 70 mg of collagenase A (Sigma, 0.235 U/mg), p.100
, mg of bovine serum albumin fraction V (BSA), 2 mL of concentrated Krebs solution
, Percoll solution is prepared by mixing 18 mL of Percoll (GE Healthcare) with 2 mL of concentrated Krebs solution. Discard 1 mL and save 19 mL of the solution for later use
, Locke's solution: 140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgSO4 , 1.2 mM KH 2 PO 4 , 10 mM EDTA, 11 mM glucose, 0.57 mM ascorbic acid, and 15 mM HEPES
, Nicotine (Sigma) is diluted first in Locke's solution at 1M and stored protected from light. This intermediate dilution can be used within few hours to prepare the final 10 µM nicotine solution in Locke's solution
, High potassium Locke's solution: Prepare Locke solution with 59 mM KCl and and NaCl
, Repeat step 3 after 10 min. Caution should be taken when performing the second injection as the gland is partially digested and may rupture with excessive pressure
, Open the gland longitudinally (along the long axis of the gland) using a scalpel and clean scissors to reveal the cream-colored medulla
, Use scalpels to collect the medulla and transfer it to a clean Petri dish. Do not collect any purple material containing cortical cells. This procedure should be performed swiftly
, Collect about 19 mL of cell suspension, add 19 mL of the Percoll solution and gently mix
, Centrifuge at 20,000 g for 20 min at 24-26°C without brake to make the self-generated Percoll gradient (13-14). Be careful to equilibrate the tubes before spinning
, Collect with a plastic Pasteur pipette the chromaffin cells in the interface of the gradient. Note that the bottom of the tubes contains red blood cells that should be discarded. Discard 2-5 mL of the top gradient containing broken and cortical cells
, Collect around 15-18 mL containing chromaffin cells and dispatch into 4 50-mL Falcon tubes
, Complete each tube with DMEM-D up to 50 mL and centrifuge 10 min at 100 g at 20°C
, Plate freshly cultured chromaffin cells in 96-well plates at a density of 50,000, vol.100, p.0
, Incubate with 50 µL of Locke's solution (resting condition) or stimulate with Locke's
, Add 50 µL of sodium hydroxyde (5M) to convert aminochromes to aminolutins
, Measure the fluorescence emitted by adrenolutin and noradrenolutin in secreted media (S) and in the cell pellets (P) (lex: 430nm, lem: 520 nm) with a spectrofluorometer. Blank values (Locke's and 0.1% Triton X-100 solutions) are subtracted to
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