Jun 25, 2024

Public workspaceMaintenance & Differentiation: Embryonic Mouse Hippocampal Cells (CLU198)

DOI
dx.doi.org/10.17504/protocols.io.bp2l628wrgqe/v1
  • Md Razaul Karim1
  • 1University of Minnesota
  • Md Razaul Karim: Lee Lab
Jane Balster
Open access
DOI: dx.doi.org/10.17504/protocols.io.bp2l628wrgqe/v1
Protocol Citation: Md Razaul Karim 2024. Maintenance & Differentiation: Embryonic Mouse Hippocampal Cells (CLU198). protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l628wrgqe/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: 102396
Keywords: Maintenance, Differentiation, Embryonic Mouse Hippocampal Cells (CLU198)
Funders Acknowledgement:
ASAP
Grant ID: 000592
Abstract
This protocol details the maintenance & differentiation of Embryonic Mouse Hippocampal Cells (CLU198).
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. Glutamax (100x) Gibco-(1x final)
III. Pen-Strep (1% final)
IV. B27-Supple (50x) Gibco-(0.5x final)

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 CLU cells, use DMEM full media.
Differentiation (CLU 198): Takes about a week of differentiation.
Differentiation (CLU 198): Takes about a week of differentiation.
33m

Note
No need to add Retinoic acid.

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.
Pipetting
Wash
Add Amount2 mL -Amount3 mL trypsin (0.25%); keep in incubator for Duration00:03:00 .
3m
Incubation
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

  • (i) Usually 1.5x10^4/ml cells for Biochem, and
  • (ii) 0.5x10^4/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.
Cells harvesting:
Cells harvesting:
20m 2s
Wash once with cold PBS.
Wash
Add cold lysis buffer.
Keep TemperatureOn ice & scrap immediately in Eppendorf tube.
Temperature
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.