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Method Article
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July 8th, 2025
Source: Bancroft, E. A., et al., Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons. J. Vis. Exp. (2020).
This video shows infecting primary mouse midbrain neurons with Adeno-Associated Viruses carrying a fluorescent calcium indicator. The indicator's fluorescence confirms successful infection and enables real-time monitoring of neuronal infection.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
NOTE: Cell culture solutions should be prepared using sterile procedures in a biological safety cabinet and filtered at 0.2 µm to prevent contamination.
1. Preparation of solutions and culture medium
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 10X NA 0.3 water-immersion objective | Olympus | UMPLFLN10XW | |
| 12 mm circular cover glass No. 1 | Phenix Research Products | MS20-121 | |
| 20X NA 0.85 oil-immersion objective | Olympus | UPLSAPO20XO | |
| 35 mm uncoated plastic cell culture dishes | VWR | 25382-348 | |
| 40X NA 0.3 water-immersion objective | Olympus | LUMPLFLN40XW | |
| 60X NA 1.35 oil-immersion objective | Olympus | UPLSAPO60XO | |
| Ampicillin (sodium) | Gold Bio | A-301-25 | |
| B-27 supplement | ThermoFisher | 17504044 | 50x stock |
| Binocular Microscope | Kent Scientific | KSCXTS-1121 | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A7030 | |
| Calcium Chloride (CaCl2), anhydrous | Sigma-Aldrich | 746495 | |
| Deoxyribonuclease I (DNase) | Sigma-Aldrich | DN25 | |
| D-glucose, andydrous | Sigma-Aldrich | RDD016 | |
| DMEM + GlutaMAX medium | ThermoFisher | 10569010 | 500 mL |
| Equine serum | ThermoFisher | 26050088 | heat-inactivated |
| Fiber Optic Illuminator, 100V | Kent Scientific | KSC5410 | |
| Filter System, PES 22UM 250ML | VWR | 28199-764 | |
| Fluoview 1000 confocal microscope | Olympus | ||
| Fluoview 1200 confocal microscope | Olympus | ||
| GlutaMAX supplement | ThermoFisher | 35050061 | |
| Hanks-balanced Salt Solution (HBSS) 1x | ThermoFisher | 14175095 | 500 mL |
| HEPES | VWR | 101170-478 | |
| HeraCell 150 CO2 incubator | Heraeus (ThermoFisher) | ||
| ImageJ v1.52e | NIH | ||
| IRIS-Fine Scissors (Round Type)-S/S Str/31*8mm/13cm | RWD | S12014-13 | |
| Kanamycin monosulfate | Gold Bio | K-120-25 | |
| Laminin | Sigma-Aldrich | L2020 | |
| L-Ascorbic acid | Sigma-Aldrich | A7506 | |
| L-glutamic acid | VWR | 97061-634 | |
| Magnesium Chloride (MgCl2), andydrous | Sigma-Aldrich | M8266 | |
| MPII Mini-Peristaltic Pump, 115/230 VAC, 50/60 Hz | Harvard Apparatus | 70-2027 | |
| MULLER Micro Forceps-Str, 0.15mm Tips, 11cm | RWD | F11014-11 | |
| NBQX | Hello Bio | HB0443 | |
| Neurobasal medium | ThermoFisher | 21103049 | 500 mL |
| Normal goat serum (NGS) | Abcam | ab7481 | |
| pAAV.Syn.GCaMP6f.WPRE.SV40 | Addgene | 100837-AAV1 | Titer: 1.00E+13 gc/ml |
| Papain | Worthington Biomedical Corporation | LS003126 | |
| Penicillin streptomycin | ThermoFisher | 15140122 | 10,000 U/mL |
| Phosphate-buffered saline (PBS) 1x | ThermoFisher | 10010049 | 500 mL |
| Poly-L-lysine | Sigma-Aldrich | P4832 | |
| Poly-L-ornithine | Sigma-Aldrich | P4957 | |
| Potassium Chloride (KCl), anhydrous | Sigma-Aldrich | 746436 | |
| Pump Head Tubing Pieces For MPII | Harvard Apparatus | 55-4148 | |
| Sodium Chloride (NaCl), anhydrous | Sigma-Aldrich | 746398 | |
| Sucrose | Sigma-Aldrich | S7903 | BioXtra, ≥99.5% (GC) |
| Time-pregnant female C57BL/6 mice | Texas A&M Institue for Genomic Medicine | ||
| Triton X-100 | Sigma-Aldrich | X100 | 500 mL |
| Wide-bore blue pipette tips P1000 | VWR | 83007-380 |
