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

Normal human oral keratinocytes were prepared from
keratinized oral epithelial tissue.



Detached oral keratyinocytes were seeded onto
collagen-treated flaks and cultured in Keratinocyte
Growth Medium (KGM)



The replication kinetics was focused on number of
normal human oral keratinocytes (NHOK)
Established primary keratinocytes from epidermis
(NHEK)









Retroviruses expressing Bmi-1
Lentivirus-based shRNA expression plasmid pLL.3-7
capable of knocking down the expression of endogenous
Bmi-1

NKOH cultures, infected with RV-Bm1 and LV-Bmi-1i,
gave more than 90% infection.
For retroviruses, selection of cell began at 48 hours after
infection with 1 μg/ml puromycin.







It’s the process of introducing nucleic acid into cells
Genetic materials, or even protein such as antibodies,
may be transfected.
The retroviruses, RV-B0 and RV-Bmi-1, were prepared
for transfection
Two days after transfection, the virus supernatant was
collected and concentrated by ultracentrifugation.






Total RNA was isolated from cultured cells and subjected
to the optional column Dna digestion to elimitate any
contaminating genome.
Real-time PCR was performed in triplicates for each
sample. A total of 45 cycles were executed.
The PCR’ s main aim is to amplify a few copies of a piece
of DNA across several orders of magnitude; the method
relies on thermal cycling.





Analytical technique which include using antibodies to
detect protein in tissue and cell by immunostaining and
enzyme-linked immunosorbent assay (ELISA).
The membrane was incubated with primary antibodies
and probed with the respective secondary antibodies.
Antibodies used: p15, p57, p53, TGF- β1, p-Smad2/3




TGFB1 is a multifunctional peptide that controls
proliferation, differentiation and other functions in many
cell types, also acts with TGFA in traformation.
To measure secreted TGF-B1 by NHOK, supernatants
with or without Bmi-1were collected, centrifuged and
stored in -80 °C.





It’s related with Luciferase activity
Luciferase is a class of oxidative enzymes used in
bioluminescence.
Luciferase con be used for the level of cellular ATP in cell
viability assays or for kinase activity assay.




NHOK were plated onto the 12- well dish with 40000
cells per well.
After 24 hours, cells treated with o without TGF-β kinase
inhibitor.
Cells collected for cell counting or changed the medium
every two days.




I. Senescing NHOK were treated with 10 ng/ml of α-TGFβ1 for 6 days
Dark green colors indicate the presence of SA β-Gal
activity, and it was observed under the microscope.
The percentages of β-Gal-positive cells were
obtained, counting in a random manner one thousands
cells.

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Normal human oral keratinocyte. how to study NHOK. Research

  • 1.
  • 2.  Normal human oral keratinocytes were prepared from keratinized oral epithelial tissue.  Detached oral keratyinocytes were seeded onto collagen-treated flaks and cultured in Keratinocyte Growth Medium (KGM)  The replication kinetics was focused on number of normal human oral keratinocytes (NHOK) Established primary keratinocytes from epidermis (NHEK) 
  • 3.     Retroviruses expressing Bmi-1 Lentivirus-based shRNA expression plasmid pLL.3-7 capable of knocking down the expression of endogenous Bmi-1 NKOH cultures, infected with RV-Bm1 and LV-Bmi-1i, gave more than 90% infection. For retroviruses, selection of cell began at 48 hours after infection with 1 μg/ml puromycin.
  • 4.     It’s the process of introducing nucleic acid into cells Genetic materials, or even protein such as antibodies, may be transfected. The retroviruses, RV-B0 and RV-Bmi-1, were prepared for transfection Two days after transfection, the virus supernatant was collected and concentrated by ultracentrifugation.
  • 5.    Total RNA was isolated from cultured cells and subjected to the optional column Dna digestion to elimitate any contaminating genome. Real-time PCR was performed in triplicates for each sample. A total of 45 cycles were executed. The PCR’ s main aim is to amplify a few copies of a piece of DNA across several orders of magnitude; the method relies on thermal cycling.
  • 6.    Analytical technique which include using antibodies to detect protein in tissue and cell by immunostaining and enzyme-linked immunosorbent assay (ELISA). The membrane was incubated with primary antibodies and probed with the respective secondary antibodies. Antibodies used: p15, p57, p53, TGF- β1, p-Smad2/3
  • 7.   TGFB1 is a multifunctional peptide that controls proliferation, differentiation and other functions in many cell types, also acts with TGFA in traformation. To measure secreted TGF-B1 by NHOK, supernatants with or without Bmi-1were collected, centrifuged and stored in -80 °C.
  • 8.    It’s related with Luciferase activity Luciferase is a class of oxidative enzymes used in bioluminescence. Luciferase con be used for the level of cellular ATP in cell viability assays or for kinase activity assay.
  • 9.    NHOK were plated onto the 12- well dish with 40000 cells per well. After 24 hours, cells treated with o without TGF-β kinase inhibitor. Cells collected for cell counting or changed the medium every two days.
  • 10.    I. Senescing NHOK were treated with 10 ng/ml of α-TGFβ1 for 6 days Dark green colors indicate the presence of SA β-Gal activity, and it was observed under the microscope. The percentages of β-Gal-positive cells were obtained, counting in a random manner one thousands cells.