Rat primary neuronal cultures were prepared from rat pup tissue at embryonic day (E) 18 containing combined cortex, hippocampus and ventricular zone. The tissue was obtained from BrainBits (Catalogue #: SDEHCV) in Hibernate-E media and used the same day for dissociation following their protocol. Briefly, tissue was incubated in a solution of Papain (BrainBits PAP) at 2 mg/mL for 30 min at 37 °C and dissociated in Hibernate-E for one minute using one sterile 9” silanized Pasteur pipette with a fire-polished tip. The cell dispersion solution was centrifuged at 214 g for 1 min, and the pellet was resuspended with 1 mL NbActiv1 (BrainBits NbActiv1 500 mL). Cell concentration was determined using a hemocytometer and neurons were plated in 12-well culture plates with 18-mm PDL-coated coverslips (Neuvitro Corporation GG-18-PDL) at a concentration of 1.3 million cells/well. Neurons were then incubated at 37 °C, 5% CO2, performing half media changes every 3-4 days with fresh NbActiv1 supplemented with PrimocinTM (InvivoGen ant-pm-1). Neurons infected with GCaMP6f as stated above were infected with AAV9-hSyn-Cre (Addgene #105553-AAV9) and AAV9-hSyn-TRPA1-myc-DIO (Salk GT3 core) at DIV4 and half media changes were performed the next day. Cultures were incubated at 37 °C, 5% CO2 until DIV10-12 and were used in electrophysiology experiments.
Nbactiv1
Manufactured by Transnetyx
NbActiv1 is a cell culture medium designed to support the growth and maintenance of neuroblasts and neuronal progenitor cells. It is a chemically-defined, serum-free, and animal component-free medium that provides the necessary nutrients and growth factors for the culture of these cell types.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Ant pm 1, by InvivoGen (1 mentions)
Nuclei buffer, by 10x Genomics (1 mentions)
Primocintm ant pm 1, by InvivoGen (1 mentions)
Ovomucoid trypsin inhibitor, by Worthington (1 mentions)
L cysteine, by Worthington (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Worthington (1 mentions)
Penicillin strep, by Thermo Fisher Scientific (1 mentions)
Dnase, by Merck Group (1 mentions)
Nbactiv4, by Transnetyx (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Matrigel coated plate, by Corning (1 mentions)
Insulin, by Merck Group (1 mentions)
7 protocols using nbactiv1
1
Rat Primary Neuronal Culture Protocol
2
Dissociation of Hippocampus and Prefrontal Cortex for 10x Genomics
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Following recommendations from 10x Genomics (Cat#CG00055 Rev C) dissected hippocampus and prefrontal cortex tissue was placed into 2.5 ml Hibernate E/B27/GlutaMax medium (BrainBits cat#HEB) at Room Temp until all samples were dissected. HEB medium was removed and replaced with 2 ml of 2 mg/ml activated papain (BrainBits cat#PAP) then incubated for 25 min at 37 °C with gentle mixing. After allowing tissue to settle, papain was removed and replaced with 2 mL fresh HEB medium and tissue was gently triturated 15–20 times using a wide-bore pipette tip and tissue left to settle. Supernatant was taken and filtered using a 30 μm cell strainer (Miltenyi Biotec cat#130-041-407) into a collection tube. To the remaining tissue, another 2 ml of fresh HEB medium was added and then triturated with a regular 1 ml pipette tip an additional 10–15 times until tissue was completely disassociated. Supernatant was taken and filtered through a 30 um cell strainer and added to the collection tube. Supernatant was then centrifuged at 400rcf for 2 min. The cell pellet was re-suspended in 1–3 ml of neuronal culture medium Nbactiv1 (BrainBits cat#Nbactiv1) depending on cell pellet size, filtered through a 30 μm cell strainer (Miltenyi Biotec cat#130-041-407), and was subsequently diluted to 1500 cells/μl in Nbactiv1 for capture on the 10x Genomics Chromium controller.
3
Single-cell ATAC-seq from tissue
4
Rat Primary Neuronal Culture Protocol
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Rat primary neuronal cultures were prepared from rat pup tissue at embryonic days (E) 18 containing combined cortex, hippocampus, and ventricular zone. The tissue was obtained from BrainBits (Catalog #: SDEHCV) in Hibernate‐E media and used the same day for dissociation following their protocol.
Briefly, tissue was incubated in a solution of papain (BrainBits PAP) at 2 mg mL−` for 30 min at 37 °C and dissociated in Hibernate‐E for 1 min using one sterile 9” silanized Pasteur pipette with a fire‐polished tip. The cell dispersion solution was centrifuged at 1100 rpm for 1 min, and the pellet was resuspended with 1 mL NbActiv1 (BrainBits NbActiv1 500 mL). The cell concentration was determined using a haemocytometer (TC20, Bio‐Rad Labs, Hercules, California, USA) and neurons were plated in 12‐well culture plates with 18‐mm PDL‐coated coverslips (GG‐18‐PDL, Neuvitro Corporation, Vancouver, Washington, USA) at a concentration of 1.3 million cells per well. Neurons were then incubated at 37 °C, 5% CO2, performing half media changes every 3–4 days with fresh NbActiv1 supplemented with PrimocinTM (ant‐pm‐1, InvivoGen, San Diego, California, USA). Cultures were incubated at 37 °C, 5% CO2 until day 10–12 and were used in DHM imaging experiments.
Briefly, tissue was incubated in a solution of papain (BrainBits PAP) at 2 mg mL−` for 30 min at 37 °C and dissociated in Hibernate‐E for 1 min using one sterile 9” silanized Pasteur pipette with a fire‐polished tip. The cell dispersion solution was centrifuged at 1100 rpm for 1 min, and the pellet was resuspended with 1 mL NbActiv1 (BrainBits NbActiv1 500 mL). The cell concentration was determined using a haemocytometer (TC20, Bio‐Rad Labs, Hercules, California, USA) and neurons were plated in 12‐well culture plates with 18‐mm PDL‐coated coverslips (GG‐18‐PDL, Neuvitro Corporation, Vancouver, Washington, USA) at a concentration of 1.3 million cells per well. Neurons were then incubated at 37 °C, 5% CO2, performing half media changes every 3–4 days with fresh NbActiv1 supplemented with PrimocinTM (ant‐pm‐1, InvivoGen, San Diego, California, USA). Cultures were incubated at 37 °C, 5% CO2 until day 10–12 and were used in DHM imaging experiments.
5
Rat Hippocampal Neuronal Culture Protocol
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Procedures for rat neuronal culture were reviewed and approved for use by the Broad Institutional Animal Care and Use Committee. For hippocampal neuronal cultures, 8–12 Embryonic Day 18 embryos were collected from each pregnant Sprague Dawley rats killed by CO2 (Taconic) processed separately. Embryo hippocampi were dissected in 4°C Hibernate E supplemented with 2% B27 supplements and 100 U/ml penicillin/strep (Thermo Fisher Scientific). Hippocampal tissues were digested in Hibernate E containing 20 U/ml papain, 1 mm L-cysteine, 0.5 mm EDTA (Worthington Biochem), and 0.01% DNase (Sigma-Aldrich) for 8 min and stopped with 0.5% ovomucoid trypsin inhibitor (Worthington Biochem) and 0.5% bovine serum albumin (BSA; Sigma-Aldrich). Neurons were plated at a density of 15,000 cells/well onto poly-d -lysine-coated, black-walled, thin-bottomed 96-well plates (Greiner Bio-One). Neurons were seeded and maintained in NbActiv1 (BrainBits) at 37°C in 95% air with a 5% CO2 humidified incubator for 19 d before use. TTX-treated neurons were treated on day 19 with 1 μm TTX (Tocris Bioscience) for 48 h then harvested for immunostaining. All procedures involving animals were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
6
Murine Neuronal Cell Culture Protocol
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Neuro-2a cells (ATCC CCL-131), from a Mus musculus neuroblastoma, were cultured as described previously (12 (link)) with the exception that coverslips were coated with poly-d -lysine (Sigma-Aldrich) followed by assay 1 day after plating at ∼70% confluence. E18 rat cortices from Sprague Dawley rats (BrainBits, LLC) were triturated to single cells as described by the supplier and plated in NBActiv4 (BrainBits, LLC) (45,000 cells/well) on glass-bottom total internal reflection (TIRF) plates (MatTek). TIRF plates were precoated with 20 μg/ml poly-d -lysine (Sigma-Aldrich) overnight, followed by 3 μg/ml mouse laminin for 3 h, and equilibrated with neurobasal medium for 30 min before plating cells in NBActiv4. Neurons were cultured for 7 to 12 days with a half-fresh media change, using NBActiv1 (BrainBits, LLC) on days 4 and 7 postplating.
7
CAPN3 Detection and Myotube Fusion Assay
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For CAPN3 detection: The protocol was adapted from Guo et al.41 (link) Cells were seeded on Matrigel-coated plates (Corning) in SMCGM with a density of 75 cells/mm2. When the cells reached confluency, myotube differentiation was induced by changing the medium to differentiation medium 1 (Dulbecco’s modified Eagle’s medium with 10 μg/mL insulin [Merck, Darmstadt, Germany], 500 μg/mL bovine serum albumin [BSA] [Merck], 10 ng/mL epidermal growth factor [Thermo Fisher Scientific], 50 μg/mL gentamycin [Merck]) for 7 days with ½ medium change every second day, followed by ½ medium change with differentiation medium 2 (Dif2) (Table S7 ) supplemented with 1 × G5 (Thermo Fisher Scientific) for 2 days. Afterward the medium was changed ½ with Dif2 (Table S7 ) without G5 for 2 days followed by 7 days in NbActiv1 (BrainBits, Springfield, IL) with ½ medium change every other day. Mature, multi-nucleated myotubes were used for downstream applications. For fusion index: cells were seeded in 8-well μ-slides with 12,000 cells/cm2. Upon reaching confluency, myotube fusion was induced by adding OptiMEM (Thermo Fisher Scientific) for 5–7 days. Cells were subjected to immunostaining as described below with antibodies in Table S8 ; ≥450 nuclei were counted per sample. To assess the fusion index the percentage of nuclei within myotubes (stained with MyHC) versus the total number of nuclei was calculated.
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