H69AR

The multiple drug resistant cell line H69AR was established from NCI-H69 cells (ATCC HTB-119) that were grown in the presence of increasing concentrations of adriamycin (doxorubicin) over a total of 14 months.

The H69AR cell line is approximately 50-fold resistant to adriamycin as compared to the parental NCI-H69 cell line.

The monoclonal antibodies QCRL-1 (ATCC HB-11765) and QCRL-3 (ATCC HB-11766) will react with fixed H69AR cells.

The cell line is cross-resistant to anthracycline analogues including daunomycin, epirubicin, menogaril, and mitoxantrone as well as to acivicin, etoposide, gramicidin D, colchicine, and the Vinca alkaloids, vincristine and vinblastine.

The cells display little or no cross-resistance to bleomycin, 5-fluorouracil, and carboplatin. They have a slight collateral sensitivity to 1-dehydrotestosterone and lidocaine.

The antibodies reacts with MRP (multidrug resistance protein), a 190,000 integral membrane phosphoglycoprotein that is overexpressed in some drug-selected resistant cell lines

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. It is recommended that the cryoprotective agent be removed immediately.  Centrifuge the cell suspension at approximately 125 x g for 5 to 10 minutes.  Discard the supernatant and resuspend the cell pellet in an appropriate amount of fresh growth medium.
4. Transfer the vial contents to an appropriate size vessel.  It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.  Cells that are difficult to detach may be placed at 37^oC to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
5. Add appropriate aliquots of the cell suspension into new culture vessels.
6. Incubate cultures at 37^oC.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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