Protein Extraction from Neuronal Cells

Protocolo

1. Cell Line Preparation

1. Thaw approximately 1,000,000 mouse neuroblastoma spinal cord cells (NSC-34 cells) that were stored in a liquid nitrogen container. Use a 37 °C water incubator until the cells are fully thawed.
2. Add fresh, warm medium (DMEM medium containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% pen-strep antibiotics).
3. Centrifuge thawed cells (SL16R) at 500 x g for 5 min, forming a cell pellet. Discard the medium and add 1 mL of new medium for the cell resuspension.
4. Place the resuspended cells in a 15 mL flask and add an extra 5 mL of medium. Incubate the cells overnight in a sterile incubator with 5% CO₂ at 37 °C.
5. In parallel, place 8 uncoated coverslips (cover glass: 12 mm, 0.13-0.17 mm thick) in a 60-mm culture dish and sterilize using 70% ethanol and exposure to 30 min of UV light until it is dry. Add medium to each dish and dispose to wash off the residual ethanol.
6. Add trypsin buffer (2.5 g of trypsin and 0.2 g of EDTA in 1 L of Hanks' Balanced Salt Solution) to the cells after they reach approximately 90% confluence (roughly 6 million cells per flask) to allow adherent cells to dissociate.
   NOTE: NSC-34 cells are sensitive and only require 15 s of trypsin buffer incubation and 1 additional minute of incubation free of trypsin before dissociation using the medium.
7. Plate the cells onto the 60 mm culture dishes already containing the coverslips with 3 mL of medium, approximately 350,000 per dish. Leave them in the incubator for 24 h to allow cell attachment onto the coverslips.

2. Protein Expression Validation

1. Place 60 mm culture dishes containing the residual cells (not used in the above experiment) from each experimental group on ice.
2. Dispose of the medium and wash the cells twice with PBS to dilute and wash the remaining medium.
3. Treat the cells with 200-400 µL of lysis buffer containing PBS, 0.5% Triton, and protease inhibitor cocktail tablet (PI). Scrape using a cell scraper.
4. Collect the cells in lysis buffer in a microcentrifuge tube and homogenize with a homogenizer at 4,000 rpm for 1 min under ice.
5. Centrifuge the homogenized cells at 2,500 x g for 10 min. Collect the supernatant, quantify the protein concentration using a Bradford assay, and store at -20 °C until use.
6. Collect between 30 and 70 µg of total protein (depending on the protein) and load it into an SDS-PAGE gel to test for protein expression using an immunoblot assay.

Materiais

Name	Company	Catalog Number	Comments
Mouse Motor Neuron-Like hybrid Cell Line (NSC-34)	tebu-bio	CLU140-A
DMEM 4.5g/L L-glucose	Biological Industries	01-055-1A
FBS European Grade	Biological Industries	04-007-1A
SL 16R Centrifuge	Thermo Scientific	75004030
Thermo Forma Series ii Water Jacketed CO2 Incubator	Thermo Scientific	3111
Cover glass 12mm dia. thick 0.13-0.17mm	Bar Naor LTD
Axiovert 100 inverted microscope	Zeiss
Leptomycin B	Sigma	L2913
PBS	Biological Industries	02-023-1A
Homogenizer motor/control/chuck/90-degree clamp	Glas-Col	099C K5424CE
MicroCL 17R Centrifuge, Refrigerated	Thermo Scientific	75002455