Making up primary cell culture stock aliquots

05/03/15

Media Supplements:
Hydrocortisone (H0888 Sigma)
1g stored at room. temp.
Weigh out approx. 10mg into an eppendorf tube and dissolve in 1ml of absolute ethanol.
Transfer to 50ml tube.
Wash out eppendorf with DMEM:F12 and top up to 20ml with same to give a conc. of
0.5mg/ml.
Filter through 0.22 filter and aliquot in 500l aliquots to add to 500ml to give working strength
of 0.5g/ml.
Store aliquots at -20C.
(Eg. 02/09/11. Weighed out 11.8 mg in 2ml eppendorf tube on microbalance. Dissolved in
1ml absolute ETOH. Transferred to 50ml tube. Washed out eppendorf and topped up to
23.6ml.)

Apo-transferrin (T1147 Sigma)
100mg stored at 4C.
Dissolve whole amount in 10ml DMEM:F12 by adding medium directly to bottle.
Filter sterilise. This gives a stock concentration of 10mg/ml. Aliquot in 500l aliquots to add
to 500ml to give working conc. of 10g/ml.
Store aliquots at -20C.

Human Insulin (I9278 Sigma)

5ml solution at 10mg/ml stored at 4C.
Add 250l to 500ml medium to give working conc of 5g/ml.

Human EGF (E9644 Sigma)

200g human recombinant EGF. Lyophilised from PBS. Stored at -20C.
[NOTE: We previously used to dissolve the EGF in DMEM:F12 containing 10% FBS (Sigma
suggested 0.1 to 1% HSA or BSA or 1-10% FBS). But this was when it was lyophilised in
acetic acid . Now it is in PBS so need to add acetic acid. However, decided to still include
FBS at 1%.]

So need to make up 2 solutions before reconstituting EGF:

1. 10mM acetic acid by adding 6l glacial acetic acid to10ml sterile water.
Add 100l FBS (1%) and filter through 0.22 filter.
2. 20ml DMEM:F12 + 200l FBS (1%).
Filter through 0.22 filter.
Resuspend EGF by adding 1ml of 10mM acetic acid directly to tube and transfer to 50ml
tube. (Probably should use a syringe!)
Rinse out original tube several times with DMEM:F12/1%FBS.
Top up to 20ml with same. This is 10g/ml stock.
Do not filter EGF solution.
Aliquot in 500l aliquots to add to 500ml medium to give working conc. of 10ng/ml.
Store aliquots at -20C.

Bovine pituitary extract (13028-014 Invitrogen)

25/26 mg in 2ml = 12.5/13mg/ml.
Thaw and aliquot into 100l and 200l aliquots.
Store at -20C.
Add 200l to 50ml HuMEC medium.
Final concentration = 50/52g/ml.

Fungizone (15290-026 Invitrogen)

250g/ml solution stored at -20C.
Thaw and aliquot in 5ml aliquots and re-freeze.
Use 10ml in 500ml (5g/ml) for organoid and cell isolation and P0 tissue culture of epithelial
cells and fibroblasts.
Use 5ml in 500ml (2.5g/ml) for P1+ epithelial cells and fibroblasts.
The myoepithelial cell medium is usually made up in 50ml lots.
To this add 500l (2.5g/ml) for P0 myos, 250l (1.25g/ml) for P1 myos and 100l
(0.5g/ml) for P2+myos.
To convert from 2.5g/ml to 5g/ml in 50ml of medium add 0.5ml.

Gentamicin (G1397 Sigma)

10ml solution stored at 4C
Use 10l in 50ml (10g/ml) for P1+ myos and 20l in 50ml (20g/ml) for the P0 myos.

FBS, pen/strep and glutamine (General lab: PAA)

Thaw bottles and aliquot in 25ml and 50ml (FBS) or 5ml (pen/strep/glu) aliquots to add to
500ml of basal medium.
All aliquots frozen at -20C.

BCM
Make up 500ml of BCM by adding 5ml pen/strep, 5 or 10ml Fungizone, 50ml FBS, 500l
hydrocortisone, 500l apo-transferrin, 500l EGF, 250l insulin to 500ml DMEM:F12 (D8437
Sigma, already contains glutamine).

MEGM
Make up 50ml of MEGM at a time because it is only used for myos and the base medium is
expensive. To 50ml of HuMEC (Invitrogen, 12753-018) add 50l hydrocortisone and EGF,
25l insulin and 200l BPE. For P0 myos add 500l Fungizone and 20l Gentamicin. For
P1 myos add 250l Fungizone and 10l Gentamicin. For P2+ myos add 100l Fungizone
and 10l Gentamicin.

So, in summary, the same concentration of hydrocortisone, insulin and EGF are used
in HuMEC for the myos and in DMEM:F12 for the epis and fibs. Then its + 10% FBS
for epis and fibs (BCM) and + BPE for myos (MEGM).

Enzymes:

DNase (10104159001 Boehringer/Roche Diagnostics).

100mg (200,000 units) stored at 4C.
Dissolve in 10ml sterile water and filter through 0.22 filter to give stock conc. of 10mg/ml.
Aliquot in 1ml, 500l, 200l and 100l aliquots and store aliquots at -20C.
Larger aliquots to be used in T/E for cell isolation at final conc of 0.4mg/ml (=0.4ml in 10ml).
Smaller aliquots to be used to prevent cell clumping 10l = 100g.

Trypsin/EDTA (Hyclone SV30031.01 Fisher Scientific)

0.25% trypsin plus 0.1% EDTA in PBS without calcium and magnesium.

Thaw and aliquot in 5ml aliquots.

Store at -20C.
Dilute this 1/5 in PBS w/o ca/mg (D8537 Sigma) to give a final concentration of 0.05%
trypsin and 0.02% EDTA.
The PAA T/E that the main TC lab uses is a 10x stock which if you dilute 1/10 would give
you the same final concentrations.

Shopping list:
PBS w/o calcium and magnesium
PBS + calcium and magnesium
DMSO
RPMI 1640 with 25mM Hepes
(NaHCO3, w/o glutamine)
DMEM:F12 with 15mM Hepes
(NaHCO3 + L-glutamine)
HuMEC basal serum free medium
Fungizone
(Amphotericin B)
Gentamicin
Hydrocortisone
Human EGF
Apo-transferrin
Human insulin
Bovine pituitary extract
Epithelial Enrich beads

Sigma D8537
Sigma D8662
Sigma D2650
Sigma R5886

14.14 for 6x 500ml

15.83 for 6x 500ml
11.71 for 5 x 5ml
16.33 for 6 x 500ml

Sigma D8437

24.97 for 6 x 500ml

Invitrogen 12753-018 52.51 for 1 x 500ml

Invitrogen 15290-026
Sigma G1397
Sigma H0888 1g
Sigma E9644 0.2mg
Sigma T1147 100mg
Sigma I9278 5ml
Invitrogen 13028-014 31.20 for 25mg
Invitrogen 161.02