Cord blood mast cells [CBMC] purification
1. Take out ficoll-paque to equilibrate at RT. Work in a darkened
chamber or with aluminum foil
2. Prepare MEM:
FCS, heat inactivated [30mins at 56°c]
Pen/Strep
Ribo/Dex mix
L-Glutamine if necessary
Thaw all ingredients in 37°c bath until liquid. Fill half of a 0.2 vecuum
filter with
MEM, then all other reagents. Filter and return to bottle, label
accordingly.
3. Open small tube in blood-container using scissors [cleaned with EtOH
70%], pour gently to a flask.
4. Add an equal volume of Hank’s
5. Prepare 415ml ficoll in falcons, wrap in foil
6. Prepare 435ml Hank’s in falcons
7. Divide the diluted blood to 4 [leave 1-2ml to balance], thrust the blood
very gently and slowly upon the ficoll. Do not mix!
8. Balance and centrifuge 25min, 1400rpm, break 0, at RT
9. The gradient consists of: [bottom to top] RBCs, ficoll, mononuclear
disc, plasma.
10. The mononuclear disc is at about 15ml height. Suck out all the plasma
until 5ml above the disc, then take all the volume from 20ml to 10ml
with a pasteur pipette, pour into a falcon with 35ml Hank’s
11. Centrifuge 7min, 1250rpm at 4°c
12. Discard sup, fill each falcon with 10ml Hank’s and resuspend. Transfer
suspension from all 4 falcons into a fresh falcon, fill to 50ml with Hank’s
13. Centrifuge as in stage 11
14. Discard sup. Resuspend pellet 2-3 times gently with 10ml MEM, then
fill to 50ml
15. Fill a second falcon with MEM, add
SCF (100 ng/ml), IL-6 (10 ng/ml) (PeproTech), and PGE2 (0.3 µM)
(Sigma-Aldrich)
Mix both falcons in a flask, split to 5 flasks 20ml in each.