hTERT RPE-1

The hTERT-immortalized retinal pigment epithelial cell line, hTERT RPE-1, was derived by transfecting the RPE-340 cell line with the pGRN145 hTERT-expressing plasmid (ATCC MBA-141). 

Cells were cultured in medium containing hygromycin B until stable clones were selected [Pubmed: 9454332, 9916802].

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.

• fetal bovine serum to a final concentration of 10%
• 0.01 mg/ml hygromycin B

1. Prepare a 25 cm² or a 75 cm² culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
2. 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).
3. 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 operations from this point on should be carried out under strict aseptic conditions.
4. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
5. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
6. Incubate the culture at 37°C in a suitable incubator.
7. A 5% CO₂/95% air atmosphere is recommended if using the medium described on this product sheet.

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005

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