Biomedical Engineering Reference
In-Depth Information
BSA (Sigma-Aldrich) and 0.25% Triton X-100 (Sigma-Aldrich) in
PBS. After removal of the blocking reagent, the cells were incubated
with polyclonal antibodies against vimentin (1:100 dilution; Santa
Cruz Biotechonology), cytokeratin 14 (AE1/AE3) (1:200 dilution;
Chemicon International), type I collagen (1:1500 dilution; Rockland
Immunochemicals),
-smooth muscle actin (Lab Vision), or CD90
(Thy-1) (1:100 dilutions; BD Pharmingen) for two hours at room
temperature. The cells were washed three times with PBS and incu-
bated with Alexa Fluor
R
secondary antibodies 466 mouse antihu-
man IgG (Invitrogen) or Alexa Fluor
R
546 rabbit antihuman IgG
(Invitrogen) for one hour at room temperature. After three washes
with PBS, the cells were counterstained using a Vectashield mount-
ingmediumwith4
,6-diamidino-2-phenylindole(DAPI)(VectorLab-
oratories). Digital images were acquired using DP Controller 1.2.1
and DP Manager 1.2.1 (Olympus).
α
35.2.3
Medium and Autologous Serum Preparation
Autologous patient serum was prepared from 100-150 mL of
peripheral blood. Peripheral blood was kept at room temperature
foronehourandthencentrifugedat4
◦
C.Thesupernatantwastrans-
ferredtoafreshcentrifugetubeandcentrifugedagaintoremoveany
remainingbloodcells.Theresultingsupernatantwassterile-filtered
using a 0.22
μ
m pore tube top filter (Corning). The filtered serum
was added to the culture medium containing antibiotics to a final
serum concentration of 10%.
35.2.4
Preparation of Cell Suspension and HA Admixture
Gingival fibroblasts were harvested from 8-12 tissue T-225 culture
flasksafterbeingwashedwithPBSandtreatedwithTrypLEExpress
(37
◦
C, 5 min). The cells were washed twice with sterile saline
(Otsuka Seiyaku, Tokyo, Japan), and resuspended in sterile saline
to a final concentration of 1.0
×
10
7
cells/mL. The cell suspension
andHA(1%,sodiumhyaluronate,ARTZ
R
,KakenPharmaceuticalCo.
Ltd, Tokyo, Japan) wasstored in 1.0 mL syringesuntil use.
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