TITLE: Maintenance &amp; Differentiation: SHSY-5Y Neuroblastoma Cells
AUTHORS: Md Razaul Karim

[DESCRIPTION]
This protocol details the maintenance & differentiation of SHSY-5Y Neuroblastoma Cells.

[STEPS]
SECTION: Maintenance
1. For regular maintenance of SHSY-5Y cells, use DMEM full media.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
2. 
 

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

Warm DMEM full media, PBS, and Trypsin in the 37 °C bead bath for 30 min. Clean the working area by using 70% ethanol.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
2.1. Sup out old media without touching cells.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
2.2. Wash by adding 5 mL PBS slowly, rinse, and rock back and forth.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
2.3. Add 2 mL-3 mL trypsin (0.25%); keep in incubator for 3 min.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
2.4. Check under microscope if cells are detached, add 5 mL media and transfer to a tube.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
2.5. Spin  .
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
2.6. Sup out and add 10 mL fresh media & re-suspend cells gently and carefully.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
2.7. 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.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
3. Day-02 (Tue): 

Replace with Complete Neurobasal Media (Without Glutamax).

 

SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
4. Day-03 (Wed): 

Rest.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
5. Day-04 (Thu):

 Rest.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
6. Day-05 (Fri):

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

 

SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
7. Day-06 (Sat): 

Rest.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
8. Day-07 (Sun): 

Rest.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
9. Day-08 (Mon): 

Replace with Complete Neurobasal Media (No Retinoic acid)/Drug treat.
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
10. Day-09 (Tue):

Drug treat if necessary /Harvesting. 
SECTION: Differentiation (SHSY-5Y) Cells: About 5-7 days differentiation. No need Glutamax
11. Day-10 (Wed): 

Drug treat if necessary /Harvesting.
SECTION: Cells harvesting:
12. Wash once with cold PBS.	
SECTION: Cells harvesting:
13. Add cold lysis buffer.
SECTION: Cells harvesting:
14. Keep on ice & scrap immediately in Eppendorf tube.
SECTION: Cells harvesting:
15. Sonicate (10 S on 2 s off 20% Amplitude, 2 Pulses)
SECTION: Cells harvesting:
16. Boil (100 °C, 10 min).
SECTION: Cells harvesting:
17. Centrifuge 13.000 rpm, 10 min, 4 °C/ Collect sup.
SECTION: Cells harvesting:
18. Keep in -80 °C Freezer.
SECTION: Cells harvesting:
19. BCA to measure protein concentration.
SECTION: Cells harvesting:
20. Prepare with sample buffer and run WB analysis.