16HBE14o- cell line人支气管上皮细胞株 BioVector NTCC质粒载体菌种细胞基因保藏中心

16HBE14o- cell line人支气管上皮细胞株

16HBE14o- Human Bronchial Epithelial Cell Line
Cat No.: NTCC925569

Name: NTCC® 16HBE14o- Human Bronchial Epithelial Cell Line
Alternate Names
16HBE
16-HBE
16HBEo-
16-HBEo
16-HBE14o

Background Information
NTCC® 16HBE14o- is a human bronchial epithelial cell line isolated from a 1-year old male heart-lung patient and immortalized with the origin-of-replication defective SV40 plasmid (pSVori-). The cell line retains characteristic features of normal differentiated bronchial epithelial cells including a cobblestone morphology, cytokeratin expression, the ability to form tight junctions, and directional ion transport. When grown with an air/liquid interface, cilia can be detected. In contrast to most other respiratory cell lines, NTCC® 16HBE14o- expresses high levels of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein. Expression of CFTR is correlated to cAMP-dependent Cl- conductance in a variety of cells, including 16HE14o- epithelial cells.

Applications
NTCC® 16HBE14o- human bronchial epithelial cell line is widely used to model barrier function of the airway epithelium and to study respiratory ion transport as well as the function of CFTR.

Quality Assurance
• Each vial contains ≥ 1X10^6 viable cells.
• Cells are tested by PCR and are negative for HPV-16, HPV-18, Hepatitis A, C, and HIV-1 & 2 viruses as assessed by a Human Essential CLEAR panel by Charles River Animal Diagnostic Services.
• Cells are negative for mycoplasma contamination.
• Each lot of cells is genotyped by STR analysis to verify the unique identity of the cell line.











Representative Data

Figure 1. NTCC® 16HBE14o- cells one day after thaw (A). 16HBE14o expresses CFTR (B, MAB3484), cytokeratin-18 (C, Abcam AB668), pan-cytokeratin (D, MAB3412) and the tight junction protein ZO-1 (E, AB2272). NTCC® 16HBE14o- cultured on air/liquid interface retained polarity as shown by apical immunostaining of CFTR (F, green).

Protocols
Fibronectin/Collagen/BSA ECM Coating of Flasks
1. Make stock solutions of the following:
a) Human Fibronectin Stock (0.5 mg/mL): Add 10 mL of a-MEM (Sigma M2279) to the glass vial containing human fibronectin
(Sigma F2006-5MG).
b) BSA, Fraction V Stock (1 mg/mL): Weigh out 200 mg BSA (Cat. No. 126575) into a 50 mL conical tube. Resuspend BSA in 200
mL 1X PBS or 1X HBS. Sterile filter using a 0.22 mm SteriCup (Cat. No. SCGPU02RE).
c) PureCol Collagen Stock (3 mg/mL): Add 5 mL of sterile 0.01N HCL to 15 mg lyophilized collagen (Sigma 5006-15MG).

2. Prepare Fibronectin/Collagen/BSA ECM Mixture.

3. Sterile filter using a 0.22 mm SteriCup (Cat. No. SCGPU02RE). Label and store at 2-8°C when not in use.
4. Coat flasks with the Fibronectin/Collagen/BSA ECM mixture (3 mL for T25, 6 mL for T75 or 15 mL for T225 flasks). Distribute ECM mixture evenly over growth surfaces by swirling. Incubate flasks at room temperature in the hood for at least 2 hours, but no more than 24 hours.
5. Drain coating solution by standing flasks upright for 1-2 minutes. Aspirate. Coated flasks may be stored at room temperature for up to 1 month.
6. Do not rinse flask before use.

Thawing Cells
1. Do not thaw the cells until the recommended medium and ECM coated flasks are on hand.
Cells are thawed and expanded in a-MEM (Sigma Cat. No. M2279), 10% FBS (Cat. No. ES-009-B) and 2 mM L-Glutamine (Cat. No. TMS-002-C) and 1X Penicillin-Streptomycin Solution (Cat. No. TMS-AB2-C) (optional).
2. Remove the vial of frozen NTCC® 16HBE14o- cells from liquid nitrogen and incubate in a 37°C water bath. Closely monitor until the cells are completely thawed. Maximum cell viability is dependent on the rapid and complete thawing of frozen cells.
IMPORTANT: Do not vortex the cells.
3. As soon as the cells are completely thawed, disinfect the outside of the vial with 70% ethanol. Proceed immediately to the next step.
4. In a laminar flow hood, use a 1 or 2 mL pipette to transfer the cells to a sterile 15 mL conical tube. Be careful not to introduce any bubbles during the transfer process.
5. Using a 10 mL pipette, slowly add dropwise 9 mL of NTCC® 16HBE14o- Expansion Medium (Step 1 above) to the 15 mL conical tube.
IMPORTANT: Do not add the entire volume of media all at once to the cells. This may result in decreased cell viability due to osmotic shock.
6. Gently mix the cell suspension by slowly pipetting up and down twice. Be careful not to introduce any bubbles.
IMPORTANT: Do not vortex the cells.
7. Centrifuge the tube at 300 x g for 2-3 minutes to pellet the cells.
8. Decant as much of the supernatant as possible. Steps 5-8 are necessary to remove residual cryopreservative (DMSO).
9. Resuspend the cells in 10 -15 mL of NTCC® 16HBE14o- Expansion Medium.
10. Transfer the cell mixture to an ECM-coated T75 tissue culture flask.
11. Incubate the cells at 37°C in a humidified incubator with 5% CO2.
12. The next day, exchange the medium with 10-15 mL of fresh NTCC® 16HBE14o- Expansion Medium. Exchange with fresh medium every other day.

Cell Passage
Note: It is absolutely critical to use Sigma T3924 Trypsin-EDTA solution. Do not attempt to make your own Trypsin dilution from other sources as it will not work well for the cells. Cells are tightly adherent. Do not use Accutase or Accumax as these are insufficient to detach the cells.
1. Cells are ready to be passaged when they reach 90 – 95% confluency.
2. Rinse flask twice with 10 – 15 mL 1X PBS w/o Ca2+, Mg2+ (Cat. No. BSS-1006-B). Aspirate after each rinse. Note: Be sure to rinse twice to remove residual FBS as cells are very tightly adherent.
3. Add 10 mL Trypsin-EDTA solution (Sigma T3924) to the T75 flask. Swirl the flask to ensure that the Trypsin-EDTA completely covers the surface of the flask.
4. Incubate in 37°C incubator for 7-8 minutes.
5. After 7-8 minutes, take the flask out and for the next 3 minutes, tap firmly on the sides of the flask to dislodge the cells. Total trypsin incubation time = 10 minutes. Do not incubate longer than 10 minutes total.
6. Transfer the dissociated cells to a 50 mL conical tube. Add 15 mL NTCC® 16HBE14o- Expansion Medium to the flask to inactivate the trypsin and collect residual cells.
7. Centrifuge at 800-1000 rpm for 3-5 minutes.
8. After centrifugation, discard the supernatant and resuspend the cell pellet in appropriate volume for cell counting.
9. Cells may be passaged using a 1:6 to 1:10 split ratio into the appropriate ECM coated flasks.

Cryopreservation of Cells
NTCC® 16HBE14o- Human Bronchial Epithelial Cell Line may be frozen in the expansion medium plus 10% DMSO using a Nalgene slow freeze Mr. Frosty container.

Supplier来源:BioVector NTCC Inc.
TEL电话:400-800-2947
Website网址: http://www.biovector.net