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技術(shù)資料如下：
KNOCKOUTSR
Serum Replacement for Embryonic Stem Cells
Cat. No. 10828 500 mL
Storage Conditions: −5 to −20°C
Intended Use:
KNOCKOUT™ SR is used to support the growth of
undifferentiated embryonic stem (ES) cells in culture. It is
intended for laboratory research use only.
Background
Over the past several years, the number of researchers culturing
embryonic stem cells has increased dramatically. This increase
is directly related to the utility of ES cells for the study of gene
function and regulation (gene targeting studies), the production
of animal models for the study of human diseases, and cell
differentiation studies. General culture conditions are well
established (1,2) and usually require ES cells to be grown on a
mitotically-inactive feeder cell layer. The media generally
recommended is a D-MEM formulation supplemented with FBS,
L-glutamine, non-essential amino acids, β-mercaptoethanol, and
antibiotics if desired. Additionally, Leukemia Inhibitory Factor
(LIF, ESGRO™ Cat. No. 13275) is often used to aid in
maintaining the pluripotential nature of ES cells.
One of the most important parameters surrounding ES cell
culture is the maintenance of ES cells in an undifferentiated
state. Until now, FBS was the only option for nutritive
supplementation of ES cell media. Unfortunately, FBS also
contains undefined factors which can promote differentiation of
ES cells. Thus, each lot must be screened prior to use. In
addition, each lot of FBS is different from the last one, or the next
one, so screening is a continuous task. Since FBS screening is
laborious and costly, once a suitable lot has been identified, a
sufficient quantity is purchased and stored to meet laboratory
needs. This ties up both laboratory funds and storage space.
Additionally, there are issues surrounding hemoglobin and
endotoxin levels in FBS. All of the drawbacks of serumsupplemented
ES cell culture, including heat-inactivation of FBS,
can be alleviated with the use of KNOCKOUT SR, Serum
Replacement for Embryonic Stem Cells. KNOCKOUT SR is a
defined formulation that will provide you with consistent growth
conditions for your ES cell cultures. ES cells grown in
KNOCKOUT SR-supplemented media are substantially less
differentiated than those grown in FBS-supplemented media,
and germline transmission is not compromised in the least (see
Tables 1 & 2). The performance of KNOCKOUT SR is further
enhanced when used in conjunction with KNOCKOUT D-MEM
(Cat. No. 10829), a special basal medium designed specifically
for ES cell culture.
ES cells cultured for multiple passages in KNOCKOUT SR or
FBS-containing ES Cell Medium yielded the following results
when injected into host blastocysts:
Table 1: Chimeric Birth Data
KNOCKOUT™SR
Containing
ES Cell Medium
FBSContaining
ES Cell Medium
# of Blastocysts
Injected
104 32
Live Pups
(%)
20 (19%) 7 (22%)
Chimeric Pups
(%)
10 (50%) 3 (43%)
Male Chimeras
(%)
7 (70%) 1 (33%)
Table 2: Germline Transmission Data
Mice Derived from KNOCKOUT™ SR ES Cells Mice Derived from FBS ES Cells
Founder #
and sex
% agouti
coat color Germline
# agouti
pups/total
(%)
Founder #
and sex
% agouti
coat color Germline
# agouti
pups/total
(%)
1 male 100 yes 12/35 (34%) 1 male 100 yes 6/6 (100%)
2 male 100 yes 22/29 (76%) 2 female 70 no no pups
3 male 100 yes 26/29 (90%) 3 female 40 no 0/17 (0%)
4 male 95 no 0/30 (0%)
5 male 75 yes 5/28 (18%)
6 male 40 yes 1/28 (4%)
7 male 5 yes 28/29 (96%)
8 female 80 yes 7/7 (100%)
9 female 70 no 0/6 (0%)
10 female 50 not bred -
Product Description
KNOCKOUT SR is a serum-free formulation designed to directly
replace FBS in ES cell culture and provide the highest level of
performance and ease of use. DO NOT HEAT-INACTIVATE
KNOCKOUT SR. Unlike FBS, KNOCKOUT SR contains no serum
factors (i.e., complement) that require inactivation prior to use.
This product may be used for the growth and maintenance of
undifferentiated ES cells at the same FBS concentration
currently used, generally 15%. If comparing KNOCKOUT SR sideby-
side with FBS, please note that KNOCKOUT SR has been
optimized for use at about 15% (12.5 - 17.5%) and a decline in
performance may be seen when used at higher concentrations
(i.e. 30%). KNOCKOUT SR can also replace FBS in media used
for blastocyst injection, and for the cryopreservation of ES cells
at the same supplementation concentration normally used.
KNOCKOUT SR cannot be used as a replacement for FBS in
the plating of feeder cells. While the formulation contains
sufficient factors to allow plating of ES cells, fibroblasts have an
increased need for undefined attachment factors and will not
adequately attach in this formulation. However, once plated, the
feeder cell layer will remain attached to the plates when placed
into media containing KNOCKOUT SR. See instructions for use
below.
When KNOCKOUT SR is used for differentiation of ES cells into
specific cell types, supplementation with growth and/or
attachment factors may need to be empirically determined.
Precautions
1. L-glutamine supplementation is required, as L-glutamine
is not contained in KNOCKOUT SR or KNOCKOUT D-MEM.
2. KNOCKOUT SR should not be heat-inactivated.
3. KNOCKOUT SR should not be used in place of FBS to plate
feeder cells.
4. For optimal performance, minimize prolonged 37°C water
bath exposure of KNOCKOUT SR, and any media made
using this formulation (see instructions for use.)
Do Not Use This Product If:
1. Product packaging appears compromised.
2. KNOCKOUT SR appears cloudy.
Storage
1. Store KNOCKOUT SR at −5 to −20°C in the dark.
2. For optimal performance, do not subject KNOCKOUT SR to
repeated freeze/thaw cycles. We recommend no more than
two freeze/thaws for optimal product performance.
Instructions for Use
As with all cell culture media, minimize unnecessary exposure to
light. To thaw, place KNOCKOUT SR in a 37°C waterbath and
swirl occasionally. Remove promptly once thawed. For optimal
performance, do not store KNOCKOUT SR at 2 to 8°C for more
than one week. If the entire bottle will not be used within one
week of thaw date, it is strongly recommended that aliquots of
the serum replacement be stored under sterile conditions at -5 to
-20°C. Gently swirl the bottle to mix prior to aseptically aliquoting
into sterile vessels of choice. Store aliquots at -5 to -20°C and
thaw as needed.
To make ES Cell Medium, add 2-4 mM L-glutamine (Cat. No.
25030) to KNOCKOUT D-MEM or other basal media. Next, add
all other supplements as you normally would (non-essential
amino acids, β-mercaptoethanol, and antibiotics if desired) being
sure to omit FBS. Replace FBS with KNOCKOUT SR at the same
concentration that FBS is routinely used. The addition of LIF (3)
to ES cell culture media is recognized to be very beneficial for
maintaining the undifferentiated nature of ES cells. There are no
direct substitutes for LIF present in KNOCKOUT SR. If the ES
cell line being used is conditioned to supplementation with LIF,
you need to continue to add LIF. If the ES cell line is not
conditioned to LIF, we have found that optimal performance of
KNOCKOUT SR is obtained only when KNOCKOUT D-MEM is
used. Therefore, if your system does not require LIF, it is
especially important to use KNOCKOUT SR in conjunction with
KNOCKOUT SR D-MEM.
If feeder cells are required, plate as you normally would in
serum-containing medium. Allow cells to attach and spread
for a minimum of 8 hours (overnight). Aspirate supernatant. If
desired, rinse feeder cells one time with ES Cell Medium
containing KNOCKOUT SR to remove traces of FBS from the
feeder layer. Plate ES cells in the KNOCKOUT SR -containing
medium onto feeder cells. Incubate overnight. Fluid-change ES
cells as you normally would, using KNOCKOUT SR-containing ES
Cell Medium. STO cells attach to plasticware a bit less firmly
than mouse embryo fibroblasts. If using STO feeder cells, use
extra care when fluid changing cultures in order to maintain an
intact feeder layer. Note: As with all cell culture media, for
optimal performance, pre-warm ES Cell Medium just prior to use.
Avoid unnecessary prolonged incubation in 37°C water baths.
KNOCKOUT SR does not contain trypsin inhibitors. Therefore,
trypsin must be removed or inactivated when culturing ES cells
in KNOCKOUT SR-containing medium.
Routine Cell Maintenance. Trypsinize ES cells as you normally
would (we recommend 0.25% Trypsin/0.04% EDTA, Cat. No.
25200), being sure to triturate thoroughly to achieve a singlecell
suspension before quenching trypsin action. Resuspend
cells in KNOCKOUT SR-containing ES Cell Medium. Centrifuge
cells to produce a cell pellet. Aspirate supernatant to remove
trypsin-containing medium, resuspend pellet in fresh KNOCKOUT
SR-containing ES Cell Medium and replate as desired.
Trypsinizing neo-resistant ES Cell Clones. When trypsinizing
ES cell clones following electroporation and selection, it is
impractical to remove trypsin by centrifugation, as working
volumes are small. We recommend quenching trypsin activity
with soybean trypsin inhibitor (Cat. No. 17075). Make a sterile 5
mg/ml solution of soybean trypsin inhibitor in Dulbecco’s PBS.
Trypsinize clones as usual. Add one-tenth the volume of
soybean trypsin inhibitor to the trypsinized ES cells. Transfer
cells to KNOCKOUT SR-supplemented medium and replate as
desired.
Alternatively, clones may be dispersed into small cell clumps
without the use of trypsin. Pick clones into a 96-well dish
containing ~100μl of KNOCKOUT SR-supplemented medium.
With a sterile P-200, pipet triturate individual colonies until cell
clumps of just a few cells are produced. Transfer cells to larger
multi-welled dishes containing additional KNOCKOUT SR -
supplemented medium.
When transfecting, we recommend electroporating ES cells in
PBS. Cell recovery and selection following electroporation can
be accomplished using KNOCKOUT SR-containing ES Cell
Medium. We recommend using GENETICIN
® in liquid (Cat. No.
10131) or dry powder form (Cat. No. 11811) at your normal
concentration in KNOCKOUT SR-containing ES Cell Medium.
Quality Control Testing
The performance of KNOCKOUT SR is tested against an inhouse,
previously approved reference lot of KNOCKOUT SR.
References
1 Robertson, E.J., ed., Oxford: IRL Press. Teratocarcinomas
and embryonic stem cells; a practical approach (1987).
2 Hogan, B., Beddington, R., Costantini, F., Lacy E. Cold Spring
Harbor Laboratory Press. Manipulating the Mouse Embryo, second
edition (1994).
3 Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.G., Moreau, J.,
Stahl, M., Rogers, D. Nature, 336, 688 (1988).
ESGROTM is a trademark of AMRAD Corporation
For further information on this or other GIBCOTM products, contact
Technical Services at the following:
United States TECH-LINE SM : 1 800 955 6288
Canada TECH-LINE: 1 800 757 8257
Outside the U.S. and Canada, refer to the GIBCO products catalogue for
the TECH-LINE in your region.
You may also contact your Invitrogen Sales Representative or our World
Wide Web site at www.invitrogen.com.
For research use only.
CAUTION: Not intended for human or animal
diagnostic or therapeutic uses.
June 2001 Form No. 3878