Biomedical Engineering Reference
In-Depth Information
PROTOCOL 7.5
Fusion of Microcell to Recipient K562 Cells
Equipment and reagents
•
Polyethylene glycol (PEG 1500, ICN): Crystals are stored at room temperature.
•
Polypropylene 50ml sterile disposable centrifuge tubes (Corning)
•
DMEM (Gibco)
•
FBS (Wisent)
•
Penicillin/streptomycin (Invitrogen)
•
Hemacytometer (Marienfeld)
•
Erythroleukemic human transformed cell line K562 (ATCC CCL-243)
•
High-speed refrigerated centrifuge (Beckman XL-90 ultracentrifuge)
•
Culture plates (150mm and 96-well) (Corning)
Method
1 Plate 100 ml of K562 cells (seeded at 2
×
10
5
/ml) in 100mm culture plates on the day
prior to the fusion, in DMEM supplemented with 10% FBS and 1% of penicillin/
streptomycin.
2 Prepare 1 ml of 50% (w/w) PEG in serum-free DMEM, dissolve at 37
◦
C, and sterilize by
filtration.
3 Count an aliquot of recipient K562 cells and pellet 2
×
10
6
cells by centrifugation.
4 Resuspend in a volume of 10ml of DMEM medium in a 50ml tube.
5 Rinse the cell pellet once and resuspend in 10ml serum-free DMEM medium.
6 Resuspend the microcell pellet (at least 2
×
10
7
microcells) in 10 ml of serum-free
DMEM. Vigorously mix the suspension by pipetting up and down to resuspend any
clumps of cells.
7 Add the resuspended microcells to the recipient K562 cell
p
suspension and mix well. Let
the cell/microcell mixture settle for 10min at room temperature and then centrifuge at
2000
g
for 10 min.
8 Aspirate the media and slowly drip 1 ml of 50% PEG w/w while gently dispersing the
pellet for 1 min. Immediately add 1 ml of serum-free DMEM in a dropwise fashion, while
gently swirling for 1 min. Incorporate an additional 1ml of serum-free DMEM under the
same conditions.
9 Add 7 ml serum-free DMEM in a dropwise fashion, while gently swirling over 2 min.
10 Centrifuge the fusion mixture at 1000
g
for 10min and gently rinse the pellet with three
changes of serum-free DMEM. Centrifuge the fusion mixture each time.
11 Resuspend the pellet in 50 ml non-selective K562 medium, place in 150mm culture
plates and incubate for 48 h at 37
◦
C.
