35 mm petri dishes

Human embryonic kidney tsA-201 cells were grown at 5 % CO₂ and 37 °C to 80 % confluence in Dulbecco’s modified Eagle’s/F-12 medium supplemented with 10 % (v/v) foetal calf serum and 100 units/ml penicillin/streptomycin. Cells were split with trypsin/EDTA and plated on 35-mm Petri dishes (Falcon) at 30–50 % confluence ∼16 h before transfection. Subsequently, tsA-201 cells were co-transfected with complementary DNAs (cDNAs) encoding wild-type or mutant Ca_V1.2 α₁ subunits with auxiliary β_2a [29 (link)] as well as α₂-δ₁ [11 (link)] subunits and GFP to identify transfected cells.
The transfection of tsA-201 cells was performed using the FuGENE HD Transfection Reagent (Roche) following standard protocols. tsA-201 cells were used until passage number 15. No variation in channel gating related to different cell passage numbers was observed.

Slices of 350 μm thickness were made using McIlwain tissue chopper (Mickle Laboratory Eng. Co., Surrey, United Kingdom), from the hippocampi of Sprague-Dawley rats of post-natal days 7–8. Tissue cultures were then transferred to a poly-D-lysine (PDL) coated substrates. For substrate microwire recordings (s-MW), 6-well plates (Falcon) were used. MEA recordings were collected from micro-electrode arrays (MEA) (60MEA 200/30 IR-TI, Multichannel systems). For optical recordings, 35mm petri-dishes (Falcon) were used as substrates. In the case of single inserted electrode recordings (i-MW), slices were placed on glass coverslips pre-coated with PDL and cultured in 6-well plates. Slices were maintained in a humidified 37 °C incubator with 5% CO₂ on a rocking platform. A serum free culture medium consisted of Neurobasal-A/B27, 30 μg/ml gentamicin, and 0.5 mM GlutaMAX (Invitrogen). Culture media was changed twice per week. All animal use protocols were approved by the Institution Animal Care and Use Committee (IACUC) at Lehigh University and were conducted in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Flies were raised on standard cornmeal-yeast-molasses diet at 25^∘C under a 12 h light/12 h dark (LD) regimen (where Zeitgeber time (ZT) 0 is time of lights on and ZT12 is time of lights off). Light intensity was kept at ∼1500 lux. Mated males were separated 1–2 days after emergence. Aging males were kept in inverted 8 oz round bottom polypropylene bottles (Genesee Scientific, San Diego, CA, USA) on 35 mm petri dishes (BD Falcon, San Jose, CA, USA) containing 15 ml of diet. Fresh diet was provided three times a week. All experiments were completed using the w¹¹¹⁸ strain.

Human embryonic kidney (HEK293) tsA-201 cells were grown at 5% CO₂ and 37 °C to 80% confluence in Dulbecco’s modified Eagle’s/F-12 medium supplemented with 10% (v/v) fetal calf serum and 100 units/ml penicillin/streptomycin. Cells were split with accutase solution and plated on 35-mm Petri dishes (Falcon) at 60–80% confluence ~24 h before transfection. Subsequently, tsA-201 cells were co-transfected with cDNAs encoding WT or mutant Cav1.2 α1 subunits with auxiliary β3a as well as α2-δ1 [16 (link)] subunits and GFP to identify transfected cells.
The transfection of tsA-201 cells was performed using the TurboFect transfection reagent (Thermo Fisher Scientific) following standard protocols. HEK293 cells were used until passage number 26. No variation in channel gating related to different cell passage numbers was observed.
In order to avoid calcium-dependent inactivation, barium ions (20 mM) were used as a charge carrier.

Cre Recombinase Gesicles (Takara) were added to cultures according to the manufacturer’s protocol. Briefly, purified LT-HSCs or LSK cells were plated in 35 mm petri dishes (2 × 10⁵/dish, FALCON, Durham, NC) containing 1 ml of infection medium [serum-free RPMI-1640 medium plus 6 µg Polybrene (Sigma) and 10 µl Cre Recombinase Gesicles]. After 4 h incubation at 37 °C, medium was changed and cells further cultured in RPMI-1640 supplemented with 1% FBS, 100 ng/ml Thpo, 50 ng/ml SCF, and 0.3 ng/ml IL-3 at 37 °C in a humidified atmosphere containing 5% CO₂ in air for 20 h.

Purified LSK cells from BM or fetal liver (1 × 10³/dish) were plated in triplicate in 35-mm petri dishes (FALCON) containing 1 ml MethoCult GF M3434 medium (StemCell Technologies Vancouver, BC). After 10 days incubation at 37 °C in 5% CO₂ in air, CFU-GM, G, M, GEMM, and BFU-E were counted.