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, The PTLs MAACh and MAHoCH were synthesized as previously described [ 5 ]. All compounds are stored in dry DMSO at ?80 °C, at a concentration of at least 2 mM, to ensure a fi nal DMSO content ?1 % during bioconjugation
, Anhydrous DMSO as solvent for stock solutions ( see Note 4 )
, Drierite desiccant (calcium sulfate) with indicator ( see Note 4 )
, Clones for the ?3, ?4, ?2, and ?4 nAChR subtypes in a vector for expression in oocytes (e.g., pNKS2) and in a vector for mammalian cell expression (e.g., pCDNA3.1 or pIRES, see Note 5 )
, Plasmid DNA mini-and maxi-prep kits
, Site-directed mutagenesis kits
, High yield capped RNA transcription kits with the adequate promoter
,
, Xenopus laevis oocytes. Freshly prepared oocytes can be ordered directly from companies such as Ecocyte Bioscience ( see Note 6 )
, Incubator (18 °C)
, Neuro-2A cells: derived from neuroblastoma in an albino strain A mouse
, supplemented with 10 % Foetal Bovine Serum (FBS), 1 % nonessential amino-acids, 100 U/ml penicillin, 100 ?g/ml streptomycin, and 2 mM Glutamax ( see Note 8 ), Culture medium: Modifi ed Essential Medium (MEM)
, Twice concentrated HEPES buffered salt solution (HeBS 2×): 280 mM NaCl, 10 mM KCl, 1.5 mM NaH 2 PO 4 ·7H 2 O, 50 mM HEPES, 13 mM glucose. pH adjusted to 7.05-7.12 with NaOH. Filter-sterilize and store at 4 °C
, mM CaCl 2 , fi lter-sterilize and store at 4 °C
, N HCl solution. An acidic transfection medium solution (pH 6.3) helps dissolving DNA-Ca 2+ phosphate precipitates
, Culture medium ( see Note 8 )
, Transfection Medium: MEM plus 14 mM glucose
, Acidic transfection medium: transfection medium, with pH adjusted to 6.3 with HCl. Filter-sterilize and store at 4 °C. Important: readjust to pH 6.3 the day of transfection
, Oocyte ringer solution (ORI): 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM HEPES, pH 7.4 with NaOH. We usually prepare ORI from a 10× stock solution
, mM KCl, 100 ml, stored at room temperature
, Patch-clamp external solution: 140 mM NaCl, 2.8 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 10 mM HEPES, 12 mM glucose. NaOH for pH adjustment (pH 7.3), fi lter-sterilize and store at 4 °C up to a week
, Patch-clamp internal solution: 140 mM KCl, 2 mM MgCl 2 , 5 mM EGTA, 5 mM HEPES. KOH for pH adjustment
, Light source ( see Note 10 )
, Air table
, Upright microscope ( see Note 11 ) with a bright fi eld oil immersion condenser and a long working distance 40× objective
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