STR identification of NCI-H69 human small cell lung cancer cells

STR identification of NCI-H69 human small cell lung cancer cells

Cell Introduction

This cell line was established from lung tissue of a 55 year old white male. The cells are non diploid and will form colonies in soft agar, preserving the morphology and ultrastructure of small cell carcinoma as well as APUD cell characteristics. Cell aggregation and growth lead to inaccurate cell counting. The cell's cytokeratin staining is positive. The N-myc gene was amplified and expressed in both mRNA and protein. The expression level of C-myc mRNA is lower than that of protein.

There is expression of c-myb, v-fes, v-fms, c-raf 1, Ha ras, Ki ras, and N-ras mRNA. Cells are prone to form tumors with typical histological features of small cell carcinoma in nude mice. Form colonies in soft agar.

Cell characteristics

1) Source: Lung of a 55 year old white male

2) Form: Suspension growth of multicellular aggregates

3) Content:>1x106 cell count

4) Specification: Packaging in T25 bottles or 1mL cryovials

5) Purpose: For scientific research purposes only.

Transportation and storage

Dry ice transportation and recovery of viable cells

(1) Transport in 1mL cryovials packaged with dry ice. After receiving, store overnight in a -80 degree freezer and transfer to liquid nitrogen or directly resuscitate. If you find that the dry ice has evaporated completely, the cryovial cap has fallen off, is damaged, or the cells are contaminated, please contact us immediately.

(2) T25 bottles of revived surviving cells will be shipped at room temperature and processed according to the procedures for cell reception upon receipt.

Processing after cell reception

1) After receiving the cells, disinfect the bottle wall with 75% alcohol and place the T25 bottle in a 37 ℃ incubator for about 2-3 hours. If you find any damage to the culture bottle, overflow of liquid, or contamination of the cells, please take photos and contact us promptly.

2) Please confirm the cell status under a 4 or 5X microscope, and take 2-3 photos (10 ×, 20 ×) of the newly received cells, as well as one photo of the appearance of the culture bottle, for retention as a basis for the cell status upon receipt during after-sales service.

3) Adherent cells: Cells are placed in a 37 ℃ incubator for 2-3 hours, and their growth and adherence are observed under a microscope. Some adherent cells may detach and form clusters due to vibration during express delivery. If the growth density of cells observed under the microscope is below 60%, the culture medium in the culture bottle can be removed (if there are unattached cells, they need to be recovered by centrifugation and resuspended into the original culture bottle), and 6-8 mL of newly prepared Quan culture medium can be added and placed in the cell culture box for further cultivation. If the cell growth density reaches 70% -80% or more, the cells can be passaged. During the passage process, if cells shed due to transportation vibrations, they need to be recovered by centrifugation.

4) Note: The transport medium (infusion medium) cannot be used to culture cells anymore. Please use the newly prepared complete culture medium according to the instructions for cell culture conditions to culture cells. After receiving the cells, it is recommended to subculture them in a T25 culture bottle at a ratio of 1:2.

STR identification of NCI-H69 human small cell lung cancer cells

1、 Preparation of culture medium and culture cryopreservation conditions

1) Prepare RPMI-1640 (recommended iCell-0002) culture medium; High quality fetal bovine serum, 10%; Double antibody, 1%.

2) Cultivation conditions: Gas phase: air, 95%; Carbon dioxide, 5%. Temperature: 37 degrees Celsius, humidity in the incubator is 70% -80%.

3) Cryovial solution: 90% serum, 10% DMSO, ready to use and prepare.

2、 Cell processing:

1) Recovery of frozen cells:

Quickly shake and thaw the cryovial containing 1mL of cell suspension in a 37 ℃ water bath, then add it to a centrifuge tube containing 4-6mL of complete culture medium and mix well. Centrifuge at 1000RPM for 3-5 minutes, discard the supernatant, and resuspend the cells in the culture medium. Then add the cell suspension to a culture bottle (or dish) containing 6-8ml of culture medium and culture overnight at 37 ℃. The next day, observe cell growth and cell density under a microscope.

2) Cell passage: If the cell density reaches 80% -90%, passage culture can be carried out.

For suspended cells, passaging can refer to the following methods

Cells grown in suspension can be maintained in their growth state by adding Wan full culture medium to the culture bottle. Generally, the cell density is maintained at 1 × 105~1 × 106 cells/mL (different cells have different density requirements) to maintain normal cell growth. If necessary, the cell suspension can be collected in a centrifuge tube at 1000rpm and centrifuged for 5 minutes. Discard the supernatant, add 1-2mL of culture medium, resuspend and mix well. Then, divide the cell suspension into new T25 bottles at a ratio of 1:2 and add 6-8ml of new Wan full culture medium prepared according to the instructions to maintain the growth vitality of the cells. Subsequent passages should be carried out according to the actual situation at a ratio of 1:2-1:5.

3) Cell cryopreservation: It is recommended to freeze a batch of cell seeds during the first 3 generations of culture after receiving the cells for subsequent experimental use.

Taking T25 bottles as an example below;

1. When freezing cells, collect digested cells into centrifuge tubes according to the process of cell passage, and use a hemocytometer to determine the freezing density of cells. The recommended freezing density for general cells is 1 × 10 ^ 6~1 × 107 live cells/ml

Centrifuge at 1000rpm for 3-5 minutes to remove the supernatant. Resuspend the cells in the prepared cell cryopreservation solution, and distribute them into a cryopreservation tube at a concentration of 1 × 10 ^ 6~1 × 107 live cells/ml per 1ml of cryopreservation solution. Label the cells with their names, generations, dates, and other information.

3. Place the cells to be frozen in a programmed cooling box, refrigerate at -80 degrees Celsius overnight, and then transfer them to a liquid nitrogen container for storage. Simultaneously record the position of the cryovial in the liquid nitrogen container for future reference and use.