Serum based differentiation media constituents
REAGENT
T4 Serum
T6 Serum
Transferrin
Glutamine
Ascorbic
Acid
MTG
PFM
FCS thawed, stands for 3 months at 4C.
FCS thawed, heat inactivated for 45min in 56C water bath. After 15min
in water bath, swirl to mix.
Stored at 4C, never freeze! Stock at 30ul/ml.
Stored at –20C, 100X. Thaw at 37C, label with date and keep at 4C no
longer than 2 weeks.
Antioxidant. Stored at –20C, 5mg/ml. Thaw at 37C, use at 10ul/ml.
Throwaway excess after one use. Never add directly on top of
transferrin.
Make working solution: 26ul MTG + 2ml IMDM. Use 3ul/ml of this
Dilution in differentiation media
Protein free hybridoma medium II from Gibco, store at 4C. After
opening, keep no longer than 1 month.
DAY 2-5 MEDIA
REAGENT
T4 serum
Glutamine
Transferrin
MTG
Ascorbic acid
IMDM
Stock
100%
200mM (100%)
30mg/ml
1.25gm/ml
5mg/ml
1X
Working conc
15%
10uL/ml - 2mM (1%)
5uL/ml – 150ug/ml-(0.5%)
Dilute 26uL in 2ml IMDM. Then use at 3uL/ml.
10uL/ml -50ug/ml (1%)
85%
DAY 6 MEDIA
REAGENT
T6 serum
PFM
Glutamine
Transferrin
MTG
Ascorbic acid
IMDM
Stock
100%
100%
200mM (100%)
30mg/ml
1.25gm/ml
5mg/ml
1X
Working conc
15%
5%
10uL/ml - 2mM (1%)
5uL/ml – 150ug/ml-(0.5%)
Dilute 26uL in 2ml IMDM. Then use at 3uL/ml.
10uL/ml -50ug/ml (1%)
85%
METHYLCELLULOSE MIXES - BLAST COLONY MIX
REAGENT
Stock
Working
Use uL/ml
Methylcellulose
100%
55%
0.55ml/ml
(1%)
D4T conditioned 100%
20%
0.2uL/ml
media
T4 Serum
100%
10%
0.1 uL/ml
Transferrin
30mg/ml
150ug/ml
5uL/ml
Ascorbic acid
5mg/ml
25 ug /ml
5 uL/ml
Volume-10ml
5.5ml
2 ml
1 ml
50uL
100 uL
MTG
1.25mg/ml 26/2ml dilution
3uL/ml
30uL
VEGF
5 ug/ml
5 ng/ml
1 uL/ml
10 uL
IL-6
5ug/ml
10ng/ml
2 uL/ml
20 uL
Vortex at high speed to mix
Allow air bubbles to rise by spinning at 1400rpm for 5 mins or sitting at RT for 30 mins
METHYLCELLULOSE MIXES - ENDOTHELIAL MIX
REAGENT
Stock
Working
Use uL/ml
Volume-10ml
Methylcellulose
100%
55%
0.55ml/ml
5.5ml
(1%)
D4T conditioned 100%
20%
0.2uL/ml
2 ml
media
T4 Serum
100%
10%
0.1 uL/ml
1 ml
Transferrin
30mg/ml
150ug/ml
5uL/ml
50uL
Ascorbic acid
5mg/ml
25 ug /ml
5 uL/ml
100 uL
MTG
1.25mg/ml 26/2ml dilution
3uL/ml
30uL
VEGF
5 ug/ml
25 ng/ml
5 uL/ml
50 uL
bFGF
10 ug/ml
20ng/ml
2 uL/ml
20 uL
Vortex at high speed to mix
Allow air bubbles to rise by spinning at 1400rpm for 5 mins or sitting at RT for 30 mins
METHYLCELLULOSE MIXES - BIG MIX
REAGENT
Stock
Working
Methylcellulose
100%
55%
(1%)
PDS
100%
10%
PFM-II
100%
5%
Transferrin
30mg/ml
150ug / ml
Ascorbic acid
5mg/ml
25 ug /ml
MTG
1.25mg/ml 26/2ml dilution
VEGF
5 ug/ml
25 ng/ml
bFGF
10 ug/ml
20ng/ml
KL CM*
100%
2%
IL-3 CM*
100%
1%
Use uL/ml
0.55ml/ml
Volume-10ml
5.5ml
0.1ml/ml
0.05ml/ml
5uL/ml
5 uL/ml
3uL/ml
5 uL/ml
2 uL/ml
20 uL/ml
10 uL/ml
1ml
500 uL
50uL
100 uL
30uL
50 uL
20 uL
200 uL
100 uL
GM-CSF CM*
100%
1%
10 uL/ml
100 uL
M-CSF
10 ug /ml
5 ng/ml
0.5 uL/ml
5 uL
EPO
2000 U/ml
4 U/ml
2 uL/ml
20 uL
TPO CM*
100%
1%
10 uL/ml
100 uL
IL-6
5ug/ml
10ng/ml
2 uL/ml
20 uL
IL-11
5ug/ml
5 ng/ml
1 uL/ml
10 uL
CM* - Conditioned Media. Vortex at high speed to mix . Allow air bubbles to rise (Spin)