NOTICE: You are viewing a page of the openwetware wiki. Our "dewikify" feature makes a wiki page appear as a normal web page. In April 2017, this feature will GO AWAY and this URL will redirect to the source URL on our wiki. We're sorry for the inconvenience.

Contents

Culturing CB Cells (Mouse Embryonic Kidney Cells)

Background

These cell lines were made from e18.5 mouse embryonic kidneys of various pkd1, pax2 and immorto genotypes.
Refer to (Making Primary Cells from Mouse Tissue protocol) for details on how this was done.
We were aiming for epithelial cells (particularly of the collecting duct) positive for the SV40 large T "immorto" gene that were either wt, pkd1-/-, pkd1-/- pax2+/- or pax2+/- in genotype. Some epithelial-like cells from mouse embryos that were immorto positive were cloned using cloning rings and bulked up at the permissive temperature of 33 degrees C in the presence of interferon-gamma. All cells stemming from cloned cells are designated with "ex cloned". During the first passage of cells not cloned we found that the fibroblast-like cells were more sensitive to trypsinisation than the epithelial-like cells meaning they would lift off after 2-3mins whereas the epithelial-like cells required 5mins to lift. To enrich for epithelial-like cell populations we performed differential trypsinisation. The lifted fibroblast-like cells were frozen down and are designated as "fib lift or lifted cells". The epithelial-like cells enriched by differential trypsinisation are designated with "differential lift eps"
All cell lines designated with "ex 33'C" have been grown at 33 degrees C with interferon-gamma (and are immorto positive). Those without this designation have been established at 37 degrees C only.
The cell lines that have been cloned are CB163E4 (IM+ pkd1-/- pax2+/+), CB167E3 (IM+ pkd1-/- pax2+/+) and CB167E2 (IM+ pkd1-/- pax2+/-). Note that the early passages are pre-cloning and contain a mixed population of cells. Remember all cells stemming from cloned cells are designated with "ex cloned". Future cloning can be performed on any of the passage 0 cells frozen down.
Some immorto positive cells have been grown at 33'C for various analyses that are not cloned such as the wt lines.
All cells were grown on collagen coated plates unless otherwise indicated eg: by "plastic". Some cells are designated with "2nd plating". This means that 24h-48h post initial plating of the cells the non-adhered cells were replated on a new cell culture vessel surface. The cells that successfully established were plated onto plastic (no collagen coating). These cells should now be grown on collagen coated cell culture vessels.

As of October 2007 - Lana Ellis.


Grow Cells on Collagen Coated Surfaces

Refer to (Collagen Coating of Cell Culture Vessels) for protocol.


Cell Culture Media used for mouse embryonic kidney cells (with additives)

Reagent 50mL 500mL Final Concentration
DMEM/F-12 (1:1) 46.85mL
insulin-transferrin-selenium (100x) 375uL3.75mL0.75x
dexamethasone (20ug/mL) 250uL2.5mL100ng/mL
triiodotyronine (20ug/mL) 15uL150uL6ng/uL
murine EGF (0.1mg/mL) 5uL50uL10ng/mL
pen/strep 0.5mL5mL
FCS 2.5mL25mL5%


Interferon-gamma


Cell Culture Vessel Volume of stock IFN-gamma to add (1x104U/mL) Final [IFN-gamma] Media volume
24 well plate 5ul of 1/10 dilution of stock IFN-gamma10U/mL0.5mL
6 well plate 2uL10U/mL2mL
T25 flask 5uL10U/mL5mL
T75 flask 25uL10U/mL25mL
T175 flask 50uL10U/mL50mL


Trypsinisation


Freezing Down Cells

65% media
25% FCS
10% DMSO


Getting Cells up from Freeze Down


Handy Hint


Media Additive Preparation

Insulin-Transferrin-Selenium (100x)

Insulin-transferrin-selenium, 10mL, Invitrogen #41400-045
Store at 4 degrees C
Ready to use


Dexamethasone (20ug/mL)

Dexamethasone, 1mg, Sigma #D8893
Lyophilised reagent stored at 4 degrees C


Triiodotyronine (20ug/mL)

Triiodotyronine, 1mg, Sigma #T5516


Murine Epidermal Growth Factor

mEGF, 0.1mg, Sigma #E4127


Interferon-gamma, mouse recombinant

0.1mg, Sigma #I4777

After contacting Sigma technical help I identified that this batch of interferon-gamma contained not less than 1x106 units
It seems that with any new batch of IFN-gamma ordered in one would need to check this (details didn't come with IFN-gamma)