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Contents

Making Primary Cells from Mouse Tissue

Materials

Sterile Gem blades (wrap gem blades in tinfoil and autoclave)
Petri dishes
1x PBS
Ice
Sterile eppendorf tubes
Eppendorf tubes containing 1mL 1x PBS on ice - sterile (or larger tubes and PBS volume if you are making cells from large tissue samples - 15ml tubes)
Cell culture media without additives, ice cold (no FCS etc as this may interfere with collagenase type II activity)
Cell culture media with additives, ice cold and warmed aliquots (research suitable media for your tissue type)
Collagenase type II
Collagen coated cell culture vessels (a T25 flask was suitable for an embryonic mouse kidney ~0.5mg tissue and chunks of human ADPKD kidney of about 0.5mg)
Dry ice (small handful to euthanise your animal)
Dissection tools soaking in 70% EtOH
Euthanisation chamber (Cherie's bench)


Cell Culture Media used for mouse embryonic kidneys (no additives)

DMEM/F-12 (1:1) Invitrogen #113330-032, 500mL
Aliquot a working volume (usually 50mL) into 50mL tube
Store at 4 degrees C


Cell Culture Media used for mouse embryonic kidneys (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%


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)


Collagenase Type II Solution

Work out volume of collagenase type II solution required (depends on how many tissue samples and sizes you'll be harvesting)
Here's some examples below of tissue that primary cells have been successfully made from.
Any excess collagenase solution can be aliquoted and stored at -20 (avoid freeze/thawing more than 2x)

Tissue Sample Collagenase Type II Solution Average Weight of Tissue Sample Volume Collagenase/Sample Amount Collagenase/Sample
HsADPKD kidney chunks 18.7.05 1mg/mLestimate 0.5g15mL30mg/g tissue
Mouse Embryonic Kidneys example 1 1mg/mL~5mg each0.2mL40mg/g tissue
Mouse Embryonic Kidneys example 2 2mg/mL~5mg each0.1mL40mg/g tissue



Euthanising Animal


Recovering Cells from Tissue


For SV40 large T Positive Cells


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