Jun 25, 2024

Public workspaceMaintenance & Differentiation: SHSY-5Y Neuroblastoma Cells

  • Md Razaul Karim1
  • 1University of Minnesota
  • Md Razaul Karim: Lee Lab
Open access
Protocol CitationMd Razaul Karim 2024. Maintenance & Differentiation: SHSY-5Y Neuroblastoma Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj6r2lx1/v1
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
We use this protocol and it's working
Created: June 07, 2024
Last Modified: June 25, 2024
Protocol Integer ID: 102398
Keywords: Maintenance, Differentiation, SHSY-5Y Neuroblastoma Cells, Cell harvesting
Funders Acknowledgement:
asap
Grant ID: 000592
Abstract
This protocol details the maintenance & differentiation of SHSY-5Y Neuroblastoma Cells.
Materials
Media:

1. Maintenance Media: DMEM full media containing (DMEM/10% FBS/1% Pen-Strep).

2. Differentiation Media: Complete Neurobasal media containing
I. Neurobasal-A (1x) Media Gibco
II. Pen-Strep (1% final)
III. B27-Supple (50x) Gibco-(1.0x final)
IV. Retinoic Acid in DMSO (Stock: 10 mM); use10 µM (Final). 1µl of stock per 1ml media.

Note
# Add Ingredient IV Retinoic acid freshly each time

For differentiation:

  • 12 Well plate (1 ml/well)
  • 6 Well plate/35 mm dish (9.5 cm2-2 ml/well)
  • 60 mm dish (21 cm2-3.5 ml/dish)
  • 100 mm dish (56 cm2-10 ml/dish)
  • 250 ml Flask for sub-culture/maintenance (10 ml) [Tissue culture flask-Greiner bio-one- Cat.No.-658 170]
ReagentCELL CULTURE FLASK, 250 ML, 75 CM², PS, RED STANDARD SCREW CAP, CLEAR, CELLSTAR® TC, STERILE, 5 PCS.greiner bio-oneCatalog #658170

Maintenance
Maintenance
For regular maintenance of SHSY-5Y cells, use DMEM full media.
Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
3m

Note
No need Glutamax.

Day-01 (Mon): Plating with DMEM full media

  • Warm DMEM full media, PBS, and Trypsin in the Temperature37 °C bead bath for Duration00:30:00 . Clean the working area by using 70% ethanol.
30m
Temperature
Sup out old media without touching cells.
Wash by adding Amount5 mL PBS slowly, rinse, and rock back and forth.
Wash
Add Amount2 mL -Amount3 mL trypsin (0.25%); keep in incubator for Duration00:03:00 .
3m
Incubation
Pipetting
Check under microscope if cells are detached, add Amount5 mL media and transfer to a tube.
Spin Shaker300 x g, 00:03:00 .
Mix
Sup out and add Amount10 mL fresh media & re-suspend cells gently and carefully.
Count cells density and split accordingly. 15,000 cells/ml for maintenance

• Usually 1.0x10^4/ml cells for Biochem, and
• 0.5x10^4/ml cells for IF.
Day-02 (Tue):

Replace with Complete Neurobasal Media (Without Glutamax).

Note
#Add Retinoic Acid freshly

Day-03 (Wed):

Rest.
Day-04 (Thu):

Rest.
Day-05 (Fri):

Replace with Complete Neurobasal Media (Without Glutamax) /(Start drug treat if necessary)

Note
# Add Retinoic Acid freshly

Day-06 (Sat):

Rest.
Day-07 (Sun):

Rest.
Day-08 (Mon):

Replace with Complete Neurobasal Media (No Retinoic acid)/Drug treat.
Day-09 (Tue):

Drug treat if necessary /Harvesting.
Day-10 (Wed):

Drug treat if necessary /Harvesting.
Cells harvesting:
Cells harvesting:
20m 2s
Wash once with cold PBS.
Wash
Add cold lysis buffer.
Keep TemperatureOn ice & scrap immediately in Eppendorf tube.
Sonicate (10 S on Duration00:00:02 off 20% Amplitude, 2 Pulses)
2s
Boil (Temperature100 °C , Duration00:10:00 ).
10m
Temperature
Centrifuge Centrifigation13.000 rpm, 4°C, 00:10:00 / Collect sup.
10m
Centrifigation
Temperature
Keep in Temperature-80 °C Freezer.
Temperature
BCA to measure protein concentration.
Prepare with sample buffer and run WB analysis.