Irradiated bFGF/LIF Producing
Feeder Cells
Features
• Low passage, already irradiated
and tested for iPSCs and ESCs
culture
• Provided as one-shot vials. Simply
thaw and plate as feeder layers
• MEF for mouse, human fibroblasts
for human
• Expressing bFGF and LIF at
appropriate levels, no need to
supply recombinant bFGF and LIF
as medium supplement, providing
extremely high efficiency and
convenience to stem cell culturing.
Website: www.allelebiotech.com
Call: 1-800-991-RNAi/858-587-6645
(Pacific Time: 9:00AM~5:00PM)
Email: oligo@allelebiotech.com
For Technical Support:
Email:iPS@allelebiotech.com
or Research Use Only. Not for
F Diagnostic or Therapeutic Use.
Purchase does not include or carry
any right to resell or transfer this prod-
uct either as a stand-alone product or
as a component of another product.
Any use of this product other than
the permitted use without the express
written authorization of Allele Biotech
is strictly prohibited
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Irradiated bFGF-Producing Human Fibroblasts
Irradiated bFGF-Producing Human Fibroblasts
Irradiated bFGF-Producing MEFs
Irradiated bFGF-Producing MEFs
Irradiated bFGF-LIF-Producing MEFs
Irradiated bFGF-LIF-Producing MEFs
Introduction
MEF or Human Fibroblast
MEF feeder cells are good for mouse
iPSCs reprogramming or ESCs
maintenance; for human iPSCs or
ESCs, human fibroblast feeder cells
are preferred. The human fibroblast
is from foreskin, immortalized
with ectopically expressed human
telomerase and cell cycle regulators at
an early passage.
bFGF-Producing
Basic FGF (bFGF) is a 17-kDa
member of the family of heparin
binding growth factors. It is a critical
component of iPSCs or ESCs
culture medium; this growth factor is
necessary for the cells to remain in
an undifferentiated state, although the
mechanisms by which it does this are
poorly defined. Allele proudly provides
bFGF-producing MEF and Human
Fibroblast feeder cells transduced
with lentiviral vectors encoding bFGF,
which not only eliminate the needs for
added recombinant bFGF to stem cell
cultures, but also form very nice cell
lawn to serve iPSC colony formation
because of their strictly controlled
passage and growth conditions.
bFGF-LIF-Producing
Leukemia inhibitory factor (LIF) is a
20 kDa protein that is known for its
ability to inhibit the differentiation of
embryonic stem cells in mice and
contribute to stem cell selfrenewal.
It is another important component
of iPSCs or ESCs culture medium.
Human and mouse LIF proteins share
ABP-SC-BFFDH05 5vial
ABP-SC-BFFDH20 20vial
ABP-SC-BFFDM05 5vial
ABP-SC-BFFDM20 20vial
ABP-SC-BLIFM05 5vial
ABP-SC-BLIFM20 20vial
79% sequence homology and exhibit
cross-species activity. However, LIF
inhibition of stem cell differentiation
appears to be mouse-specific. That’s
why Allele only provides bFGF-LIF
producing MEF.
Cell Lines?
There are publications showing the use
of cells lines as feeder cells instead of
primary fibroblasts, e.g. SL10, MRC-
5, STO. Using cell lines would be
easier than primary cells. However,
we provide validated bFGF and LIF
producing feeder cells at prices often
lower than those from cell lines.
Drug Resistance?
There are companies offering drug-
resistant feeder cells. There has
not been any request so far for us
to include them in our catalogue.
However, for those who do need to
use drug selection for any reason, we
will provide drug-resistant feeder cells
upon request.
Fluorescence Labeling?
Some companies provide Fluorescent
protein expressing feeder cells for easy
separation from iPSCs. As feeder cells
are quite different in morphology from
iPS cells or ES cells, we do not include
fluorescent protein in feeder cells,
even though Allele is a FP technology
developing company. One way prefer
to use the limited colors of FPs in stem
cells. For details , check our Stem Cell
Reporter product line.
 Protocols
Storage: liquid nitrogen (preferred) or
-80°C
Content: 2x10^6 cells per vial
Steps:
1. Thaw one vial of irradiated feeder
cells by swirling gently in 37oC
water bath until all of the contents
are thawed. One vial of 2x10^6
cells is sufficient to prepare two10-
cm dishes, or two 6-well or 12-well
plates (about 3-4x10^4/cm2).
Note: Uncoated plates may be used,
however, depending on the brand and
quality of different type of plate, the
efficiency of attachment may vary.
We recommend first time users thaw
one vial of feeder cells and plate on
one 10 cm dish or 6 well plate to see
if the cells attach well and cover the
entire surface of the plate bottom. Cell
metabolism may cause the medium to
turn yellowish after 3 to 4 days, medium
may be changed at that time. A small
percentage of cells may come off, but
the majority of the feeder cells should
remain attached as a lawn for about
10 days to 2 weeks, during which time
stem cells may be grown on them.
2. Spray vial with 70% ethanol and
wipe dry before placing in tissue
culture hood.
3. Gently add 1 ml prewarmed
feeder cell medium (alphaMEM
or DMEM/F12 with 10% FBS),
mix with contents of cryovial and
transfer into 15-ml conical tube
containing 4 ml prewarmed feeder
cell medium.
4. Centrifuge the cells at 200g at
room temperature for 5 min and
discard the supernatant.
5. Resuspend the feeder cells in 12
ml feeder cell medium. Gently
shake the dish left/right and up/
down 10-20 times without swirling
the plate to evenly distribute the
cells across the plate.
If using a 6-well plate: add 1 ml
of feeder cell suspension to each
well of the 6-well plate containing
1 ml fresh feeder cell media per
well.
If using a 10-cm tissue culture
dish: add 6 ml of feeder cell
Allele Biotech-Introducing Cost Effectiveness to Research
suspension to 10-cm tissue
culture dish containing 6 ml fresh
feeder cell media.
If using a 12-well plate: add 0.5
ml feeder cell suspension to each
well of 12-well plate containing
1 ml fresh feeder cell media per
well.
6. Incubate the cells in 37 1C, 5%
CO2, overnight.
CRITICAL STEP When moving the
feeder cell plates from the tissue
culture hood to incubator, do not swirl
the medium, as this tends to cause
the cells to accumulate in the center.
Immediately after placing the plates in
the incubator, slide the plates forward
and backward (2–3 cm) two times,
then left to right (2–3 cm) two times to
ensure equal distribution of the cells.
Use within 5–7 days.
7. Split stem cells (~2.5 x 10^5 to 5
x 10^5 cells, or ~10% confluence)
into plate with feeder cells:
aspirate medium from ESC or
iPSC, wash with PBS and add 0.5
ml of 0.05% trypsin. Incubate at
37oC, 5% CO2, for 5 min.
8. Inactivate trypsin with 3 ml stem
cell medium (e.g. DMEM + 20%
knockout serum replacement), and
collect cell clumps in 15-ml conical
tube avoiding making single cell
suspension because ESC tends to
die in single cell form.
9. Centrifuge at 200g at room
temperature for 4 min.
10. Aspirate feeder medium from
feeder plates (cells incubated
in Step 6), rinse with one ml of
stem cell medium and add 5 ml
of stem cell medium and return to
incubator.
11. Aspirate and discard supernatant
from the conical tube in Step 8,
resuspend cells in 5 ml stem cell
medium, gently dispense the cell
pellet three times, add to feeder
cell wells or dishes.
12. Incubate stem cells grown on
feeder cells at 37oC, 5% CO2, for
48 h.
13. Aspirate medium and replace
with stem cell medium every day;
if iPSC colony number is low,
replace medium every two days.
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 Set: OSKMP
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 Pre-Mix: OSNL
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Primer Set (50 reactions
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 Antibodies (Check online)
 Irradiated bFGF/LIF
Producing Feeder Cells
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