TITLE: Maintenance &amp; Differentiation: 	Embryonic Mouse Hippocampal Cells (CLU198)
AUTHORS: Md Razaul Karim

[DESCRIPTION]
This protocol details the maintenance & differentiation of Embryonic Mouse Hippocampal Cells (CLU198).

[STEPS]
SECTION: Maintenance
1. For regular maintenance of CLU cells, use DMEM full media.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
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 (CLU 198): Takes about a week of differentiation.
2.1. Sup out old media without touching cells.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
2.2. Wash by adding 5 mL PBS slowly, rinse, and rock back and forth.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
2.3. Add 2 mL-3 mL trypsin (0.25%); keep in incubator for 3 min.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
2.4. Check under microscope if cells are detached, add 5 mL media and transfer to a tube.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
2.5. Spin  .
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
2.6. Sup out and add 10 mL fresh media & re-suspend cells gently and carefully.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
2.7. Count cells density and split accordingly. 15,000 cells/ml for maintenance

(i) Usually 1.5x10^4/ml cells for Biochem, and 
(ii) 0.5x10^4/ml cells for IF.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
3. Day-02 (Tue): 

Replace with Complete Neurobasal Media.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
4. Day-03 (Wed): 

Rest.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
5. Day-04 (Thu): 

Rest.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
6. Day-05 (Fri): 

Replace with Complete Neurobasal Media/ (Start drug treat if necessary).
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
7. Day-06 (Sat): 

Rest.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
8. Day-07 (Sun): 

Rest.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
9. Day-08 (Mon): 

Replace with Neurobasal Media/Drug treat. 
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
10. Day-09 (Tue): 

Drug treat if necessary /Harvesting.
SECTION: Differentiation (CLU 198): Takes about a week of differentiation.
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.