Jan 24, 2015
Neuro-2a cell culture (Neuro 2a, N2a)
Protocol Citation: Christopher Bartley: Neuro-2a cell culture (Neuro 2a, N2a). protocols.io https://dx.doi.org/10.17504/protocols.io.cpvvn5
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: January 23, 2015
Last Modified: March 28, 2018
Protocol Integer ID: 469
Keywords: culturing neuro2a cell, neuro2a cell, neuro 2a, n2a, cell
Abstract
Basic Protocol for culturing Neuro2a cells.
Guidelines
The following are taken directly from the vendor website, I use high glucose DMEM instead of Eagle's MEM.
Complete Growth Medium The base medium for this cell line is ATCC-formulated Eagle's Minimum Essential Medium, Catalog No. 30-2003. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Subculturing Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
Materials
MATERIALS
DMEM, High GlucoseLife TechnologiesCatalog #11965-092
Fetal Bovine Serum, qualifiedLife TechnologiesCatalog #10437-028
Neuro-2a (ATCC® CCL-131™)ATCCCatalog #CCL-131
Troubleshooting
For a T-75 flask:
Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 10 mL of complete growth medium and aspirate cells by gently pipetting.
Pipett 10mL cell suspension through 60um filter into 40mL to achieve a 1:5 split
Plate cells in appropriate vessel (T-75 flask or 6-well plate) and incubate at 37C
Change media every 2 - 4 days
Resubculture when cells are 70 - 90% confluent