Revisedprotocolforestablishingamammarycelllinefromtumors

Revised protocol for establishing a mammary cell line from tumors

Drench mouse in 70% ethanol, pin and dissect open

Sterile dissection of tumor- leave some behind to use in histology

Place tumor in small culture dish

Weigh tumor in dish (empty dish weighs 2.8g)

Cover tumor with DMEM

Dissect remainder of mouse in non-sterile conditions (usual protocol). Include samples of tumor tissue in formalin and liq N2.

Prepare trypsin mix to digest tumor:

5 ml trypsin stock in 50 ml tube and add HANKs to 50 ml total vol (1x trypsin)

Mince tumor in culture dish using sterile scalpels until in cubes approx. 1mm³

Place minced tumor into 50 ml tube with 25 ml of trypsin mix. Add 1 drop DNAse. (NB. Depending on size of tumor might want to put into 2 tubes of 25 ml)

Shake tube(s) horizontallly at 37deg 60 rpm.

Observe at 15 min shaking

Observe at 30 min shaking — retrieve?

If DNA has formed big blob of debris, add large amount (? 0.5 ml) DNAse to the blob and break it up with pipetting (10 ml pipette and / or pasteur).

Once blob sufficiently broken up, combine with remainder of cell mixture and filter through sterile gauze into sterile 50 ml tube.

Add equal volume of DMEM to cells to stop trypsin

Centrifuge 800 rpm 5 min

Decant supernatent

Resuspend pellet in 10 ml MEGM

Count cells

Prepare ECM gel:

Thaw ECM in room temp water then keep on ice

Chill few ml MEGM in 50 ml tube on ice

Chill 6 well plate on ice

Mix 1.5 ml ECM with 1.5 ml MEGM and keep mixture on ice

Pipette 600ul of mixture into the bottom of the 6 well plate — 4 wells

Allow to gel at room temp in hood

Try approx 1 million cells per well (x2)

Also 2 million cells per well (x2)

Calculate volumes of cells needed for these numbers. Dilute in prechilled MEGM to 1 ml of appropriate concentration cells

Add 1 ml of ECM to cell mixes and mix — keep chilled.

Pipette 1 ml of cells/MEGM/ECM mix to each well and mark the amount of cells added.

Allow to gel at room temp in hood

Cover with 1 ml MEGM when gelled

Incubator

Remainder of cells into 100 mm plates:

If possible set up 4 culture dishes MEGM x2

1 MEGM: 2 KGM

(One MEGM is for immunostaining; one 1 MEGM: 2 KGM will be changed to KGM only once cells have stuck down.)