For regular maintenance of CLU cells, use DMEM full media.

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

• Warm DMEM full media, PBS, and Trypsin in the 37°C bead bath for 0h 30m 0s. Clean the working area by using 70% ethanol.

Sup out old media without touching cells.

Wash by adding 5mL PBS slowly, rinse, and rock back and forth.

Add 2mL-3mL trypsin (0.25%); keep in incubator for 0h 3m 0s.

Check under microscope if cells are detached, add 5mL media and transfer to a tube.

Spin 300x g.

Sup out and add 10mL fresh media & re-suspend cells gently and carefully.

Count cells density and split accordingly. 15,000 cells/ml for maintenance

• (i) Usually 1.5 x10^4/ml cells for Biochem, and
• (ii) 0.5 x10^4/ml cells for IF.

Day-02 (Tue):

Replace with Complete Neurobasal Media.

Day-03 (Wed):

Rest.

Day-04 (Thu):

Rest.

Day-05 (Fri):

Replace with Complete Neurobasal Media/ (Start drug treat if necessary).

Day-06 (Sat):

Rest.

Day-07 (Sun):

Rest.

Day-08 (Mon):

Replace with Neurobasal Media/Drug treat.

Day-09 (Tue):

Drug treat if necessary /Harvesting.

Day-10 (Wed):

Drug treat if necessary /Harvesting.

Wash once with cold PBS.

Add cold lysis buffer.

Keep On ice & scrap immediately in Eppendorf tube.

Sonicate (10 S on 0h 0m 2s off 20% Amplitude, 2 Pulses ).

Boil (100°C, 0h 10m 0s).

Centrifuge 13.000rpm,4°C/ Collect sup.

Keep in -80°C Freezer.

BCA to measure protein concentration.

Prepare with sample buffer and run WB analysis.