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- 1419
- 威望
- 1419
- 包包
- 1887
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2. Fibroblast Growth Medium (FGM?), as either:$ g4 N; W N4 Z' D' O4 w* a$ y
Fibroblast Growth Medium BulletKit? (FGM? BulletKit?) (CC-3130), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM?) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots? (amounts indicate concentration of each SingleQuot?)
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1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml3 T9 B# p: u+ u
5 mg/ml Insulin (CC-4021) 0.5 ml9 Z/ K# z* N7 d% W
50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml$ C. {; z. B! ~7 n/ J
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(or)2 V* |; K+ |: }
Fibroblast Growth Medium-2 BulletKit? (FGM?-2 BulletKit? ) (CC-3132), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM?) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots? (amounts indicate concentration of each SingleQuot?)& y% S" a) V# \+ Z
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1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
, r, I6 M E+ d) X5 }- n2 `) o5 mg/ml Insulin (CC-4021) 0.5 ml
% F$ A: ?5 y. y3 ^& @0 i5 _50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml
( n+ p I9 v# }! Z10 ml FBS (Fetal Bovine Serum) (CC-4101)
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3. ReagentPackTM (CC-5034) contains one 100 ml bottle of each of the following subculture reagents:2 O3 u1 c g, P9 a
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HEPES Buffered Saline Solution (HEPES-BSS) (CC-5022)1 x 100 ml bottle( ?3 b' R" w* a/ n: ?
Trypsin/EDTA Solution (T/E) (CC-5012) 1 x 100 ml bottle
% d/ U+ c6 x/ B/ \) C$ vTrypsin Neutralizing Solution (TNS) (CC-5002) 1 x 100 ml bottle0 r9 }0 P7 @5 T/ K7 S" C
+ }3 m9 B7 \- R2 p4 l' {% ] k
NOTE: If you use a different Clonetics? medium, see Appendix A.
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General Information7 _+ @8 I7 l) e- h
Product Applications7 u* Y$ P& [! ?& H7 B L$ s
Clonetics? Normal Human NHDF and NHLF are:1 {. u5 Z/ U6 r
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FOR RESEARCH USE ONLY. % n! L. A# W, Z6 y( `
NOT approved for human or veterinary use, for application to humans or animals, or for in vitro diagnostic procedures.
2 D3 X! d! [' B. J* FMaterials Not Provided3 d, G9 |1 p" n* `: J1 B5 K ~& W
Fibroblast Cell Systems do not contain plasticware, glassware or other laboratory equipment used in a cell culture laboratory. Individual components are available separately.
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0 z* T/ Q( t- D/ a6 a8 W( E; s4 D9 dProduct Warranty
; N3 w0 j7 S0 Z! h% B) j* C, bCULTURES HAVE A FINITE LIFESPAN IN VITRO. BioWhittaker warrants its Clonetics? cells in the following manner only if Clonetics? media and reagents are used.
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NHDF and NHLF cryopreserved cultures are assured for experimental use for fifteen population doublings. . V9 ~9 _3 o4 P4 b8 ?8 n0 @
NHDF and NHLF proliferating cultures are assured for experimental use for ten population doublings. % k, Q; L4 j% V9 l6 y
Additional population doublings and subcultures are possible, but growth rate, biological responsiveness and function deteriorate with subsequent passage.
^3 l# O! B \8 Z; DNHDF and NHLF can become irreversibly contact-inhibited if maintained at confluence for more than two days. To avoid the loss of your cells and forfeiture of your warranty, we recommend that you subculture cells before they reach 90% confluence. . u: J: d' U! r4 F/ j& V( v2 Q
Cell Isolation . c6 o4 T) T8 Z' a( V" ^! u
Fibroblast cultures are established at BioWhittaker's cell culture facility from normal human tissue.6 [* X' T: n+ [
! M4 i( h- W7 t/ [/ i) fNHDF are isolated according to proprietary procedures, incubated until they approach 90% confluence, harvested and cryopreserved as frozen primaries. 4 S% P; W+ R* ]! \4 j
NHLF are isolated according to proprietary procedures, incubated until they reach 90% confluence, subcultured once and cryopreserved as frozen secondary cultures. 3 B* S! K) [9 s; x' b
NHDF and NHLF are cryopreserved in FGM? (or) FGM?-2 supplemented with 10% v/v fetal bovine serum and 10% v/v dimethylsulfoxide as a cryopreservation solution to improve cell viability and seeding efficiency upon thawing. 1 R) t) ~1 ] s
Medium Information
4 f! s4 R0 C% p( m! EFGM? BulletKit? (CC-3130) and FGM?-2BulletKit? (CC-3132)
6 |! L' H! \6 yHow Prepared Storage Requirements Shelf Life & |' t' U C* Y' d
All FGM ? BulletKit ? and FGM?- 2 BulletKit ? components have been human cell culture-tested. All solutions are sterile-filtered by passage through a 0.2 mm filter. Basal medium is stored at 2? to 8?C, and growth factors are stored at -20?C until shipment. If thawed upon arrival, growth factors can be stored at 2? to 8?C and added to FBM ? within 72 hours of receipt.
$ ]2 h- }( A R& e4 oIf thawed and will NOT be used within 72 hours, growth factors must be refrozen. They may be refrozen only once and then stored at -20?C for up to one year.
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! F5 F: V$ k2 [: S$ F& OStore FBM ? and FBM ?-2 at 2? to 8?C Store fully supplemented FGM ? and FGM ?-2 at 2? to 8?C.
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Avoid repeated warming and cooling of the medium. If the entire contents are not needed for a single procedure, transfer only the required volume to a sterile secondary container. Do not freeze. ( T* {5 i3 H6 C5 b: s2 M
FGM ? BulletKit ? and FGM ?-2 BulletKit ? shelf life are limited by the shelf life of the FBM ?, which is 1 year from the date of manufacture. When growth factors are added at any time within this time period, we recommend use within 1 month, but before the basal medium expiration.
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Quality Control ( E: t6 \3 j5 I7 a6 _' a! `& e
NHDF and NHLF are cultured without antimicrobial agents and assayed to ensure the absence of microbial contamination after cryopreservation.2 H& q, x4 L& N) x1 f& h8 r
3 \& X1 A* b, t2 _, C7 E7 DAll cell strains test negative by PCR(1) for HIV-1, hepatitis-B and hepatitis-C.
, G5 N9 B8 T1 b, F: A. k, h' r: R0 f6 PAfter recovery from liquid nitrogen, cells are tested for viability, growth rate, morphology, seeding efficiency, proliferative capacity, mycoplasma, yeast, fungus and bacteria. Each culture meets Clonetics? product specifications for proliferative capacity, (i.e., 15 cumulative population doublings after thaw). ! E/ M" y+ H; a. `2 y1 Z" c# G) S
Additional Testing: , J4 c6 _& |& m# M5 F& G) k
Cell Type Von Willebrand Factor Smooth Muscle Alpha Actin Cytokeratins 18 & 19
% E- l0 d9 g2 w# @NHLF Negative Negative Negative 0 D! {2 L+ }$ Y( s$ d% f) P
. H, W/ m% o% B4 T2 Y; ZBefore shipping, all basal media, growth factors and cell culture reagents are tested for sterility and cell culture performance.
, p1 b+ G+ c" r$ aSubculture Reagent Storage1 |" x. _& j$ c9 W7 U! ]4 k! I
1. Subculture reagents are stored at -20?C until shipped from BioWhittaker's Distribution Centers.
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2. Subculture reagents may thaw during transport. They may be refrozen once.8 M# p; Y$ d9 V& A' y7 z$ k
6 E) [: i8 ]7 Y- ]) ?5 Z3. Subculture reagents can be stored at -20?C for up to one year after thawing once and refreezing.
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4. To keep Trypsin/EDTA fresh and active after thawing, you should aliquot it into five 20 ml sterile centrifuge tubes and refreeze at -20?C. Trypsin/EDTA may be stored frozen up to one year.# j/ p& ^' @& y) E3 ]( o
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5. We recommend that HEPES-BSS and the Trypsin Neutralizing Solution, once stored at 4?C, be used within one month.
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' C% \( L' m% g* j$ k* u3 ? pHandling Precautions ! Q0 \+ L; }' b4 i
Normal human cells are fragile, and require special handling:
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Upon receipt, immediately store cryopreserved cells in liquid nitrogen. Properly stored cells remain viable indefinitely. 0 X) x6 g$ O6 ]8 K Q' `3 k" ^
Upon receipt, immediately place proliferating cells in a 37?C, 5% CO2, humidified incubator. 0 Y( A; B$ @+ d; ?
Do not use the medium or reagents beyond the expiration date.
& [; ]. S% q$ R" ^$ tNormal human cells are very sensitive to impurities in commercially available Trypsin. Use only Clonetics? Trypsin; every lot of our Trypsin is tested on normal human cell cultures. 5 ?6 q( x1 H9 N
Use only Clonetics? media. Keep media refrigerated at 4?C. When using a medium, take just the amount you need and then return the bottle to the refrigerator. 2 @* B& o5 g1 G" y8 R4 M
Regularly wipe flasks, cryovials, bottles and gloves with 70% isopropyl alcohol or 70% ethyl alcohol.
: z4 R: h# t" @+ G( f# |- u8 yBecause cells are anchored to one side of a flask, always add all liquids by pipeting them down the opposite side from where the cells are attached. |
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