NCI-H460, also known as H460, was derived from a male patient with large cell lung carcinoma. NCI-H460 cells are adherent cells growing twice as fast than the A549 cells with a doubling time of 33 hours in RPMI 1640 supplemented with 10% FBS. They can form tumors in both in vitro and in vivo models, including nude mice.
The cells have a hypotriploid karyotype with a modal chromosome number of 57, ranging from 53 to 65. Seven marker chromosomes are common to all cells, including der(9)t(1;9)(q21;p24), der(9)t(7;9)(p11;p22), t(10q14q), der(16)t(7;16)(q11.23;q22).
NCI-H460 cells have been shown to express p53 mRNA at high levels comparable to normal lung tissue, while exhibiting no gross structural DNA abnormalities. They stain positively for keratin and vimentin but are negative for neurofilament triplet protein. Isoenzyme analysis has shown that HPRT is localized on the surface of these non-small-cell lung cancer cell lines. AK-1, ES-D, and Me-2 isoenzymes are expressed at level 1, while G6PD and PGM1 and PGM3 isoenzymes are expressed at level B and 1-2, respectively.
In conclusion, NCI-H460 cells are an important tool for studying non-small-cell lung cancer. They are tumorigenic and have a hypotriploid karyotype with a modal chromosome number of 57. These cells have a faster growth rate than A549 cells and express several isoenzymes and proteins, including HPRT, keratin, and vimentin. Their high expression levels of p53 mRNA make them a suitable model for studying the molecular mechanisms of non-small-cell lung cancer.
Remove medium and rinse the adherent cells using PBS without calcium and magnesium (3-5 ml PBS for T25, 5-10 ml for T75 cell culture flasks). Add Accutase (1-2 ml per T25, 2.5 ml per T75 cell culture flask), the cell sheet must be covered completely. Incubate at ambient temperature for 8-10 minutes. Carefully resuspend the cells with medium (10 ml), centrifuge for 3 min at 300 g, resuspend cells in fresh medium and dispense into new flasks which contain fresh medium.
1:2 to 1:4
2 to 3 times per week
CM-1 (CLS catalog number 800100) or CM-ACF (CLS catalog number 806100)
Handling of cryopreserved cultures
The cells come deep-frozen shipped on dry ice. Please make sure that the vial is still frozen. If immediate culturing is not intended, the cryovial must be stored below -150 degree Celsius after arrival. If immediate culturing is intended, please follow the below instructions: Quickly thaw by rapid agitation in a 37 degree Celsius water bath within 40-60 seconds. The water bath should have clean water containing an antimicrobial agent. As soon as the sample has thawed, remove the cryovial from the water bath. A small ice clump should still remain and the vial should still be cold. From now on, all operations should be carried out under aseptic conditions. Transfer the cryovial to a sterile flow cabinet and wipe with 70% alcohol. Carefully open the vial and transfer the cell suspension into a 15 ml centrifuge tube containing 8 ml of culture medium (room temperature). Resuspend the cells carefully. Centrifuge at 300 x g for 3 min and discard the supernatant. The centrifugation step may be omitted, but in this case the remains of the freeze medium have to be removed 24 hours later. Resuspend the cells carefully in 10 ml fresh cell culture medium and transfer them into two T25 cell culture flasks. All further steps are described in the subculture section.
Handling of proliferating cultures
One or two cell culture flasks come filled with cell culture medium. Collect the entire medium in 1 or 2 x 50 ml centrifuge tubes, respectively. Carefully add 5 ml of cell culture medium to each T25 cell culture flask. Control the cell morphology and confluency under the microscope. Incubate at 37 degree Celsius for a minimum of 24 hours. Spin down the collected medium at 300 x g for 3 minutes to collect the cells which may have detached during transit. If a cell pellet is visible, resuspend the cells in 5 ml of cell culture medium and transfer to a T25 cell culture flask. Incubate at 37 degree Celsius for a minimum of 24 hours.
Quality control / Genetic profile / HLA
Mycoplasma contamination was excluded through PCR-based and luminescence-based mycoplasma assays. Bacterial or fungal contaminations are detected through daily visual cell monitoring.
Penta E: 5
Penta D: 11,13
To identify mycoplasma contaminations we perform PCR-based and luminescence-based mycoplasma assays. We further determine any bacterial or fungal contamination through our standardized manufacturing processes.
Besides genomic DNA, RNA, cell pellets, and cell lysates, we can offer large quantities of assay-ready cells, plated cells in multiple formats, and frozen or growing cells. Contact us to receive a quote.
Each manufactured batch of cell lines* is authenticated via STR analysis. Contact us if you require a publication-ready STR report for your cells (*human, hamster, mouse, rat, and dog cells).
HLA characterization is available from more than 200 cell lines. HLA class I -A, B, C, and Class II HLA-DPA1, -DPB1, -DQA1, DQB1, and DRB1 alleles were obtained by next-generation sequencing methodologies (NGS) for class I and class II alleles.
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