Human colorectal adenocarcinoma cell line HCT15/DDP resistant to cisplatin

Cell name:Human colorectal adenocarcinoma cell line resistant to cisplatinHCT15/DDP　

Item No.WS155（（HCT15）STR 鉴定）　　

Specifications1 × 10 ⁶ cells/T25 culture bottle

Purpose: For scientific research purposes only

1、 Cell Introduction

The cell isA DDP resistant cell line constructed from HCT15 cells.

2、 Cell characteristics

1) Source: Rectal cancer

2) Morphology: Epithelial like, adherent growth

3) Content:>1x106 cell count

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

5) Purpose: For scientific research purposes only.

3、 Preparation of culture medium and culture cryopreservation conditions

1）Please refer to the instruction manual for details.

We recommend serum FBS500-WP-002

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.

4cell processing

1) Recovery of frozen cells

Will containThe frozen tube containing 1mL cell suspension was rapidly shaken and thawed in a 37 ℃ water bath, and then added to a centrifuge tube containing 4-6mL of culture medium and mixed evenly. Centrifuge at 1000RPM for 3-5 minutes, discard the supernatant and culture mediumResurrected cells. 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 the passage of adherent cells, the following methods can be referred to

1. Discard the culture supernatant and rinse the cells 1-2 times with PBS that does not contain calcium or magnesium ions.

2. Add 0.25% (w/v) pancreatic white enzyme-0.53 mM EDTA to culture bottles (T25 bottle 1-2mL, T75 bottle 2-3mL), place in a 37 ℃ incubator for digestion for 1-2 minutes (difficult to digest cells can extend the digestion time appropriately), and then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take them back to the operating table, tap the culture bottle a few times, and add 3-4ml of culture medium containing 10% FBS to terminate digestion.

3. Gently mix and aspirate, centrifuge at 1000RPM for 3-5 minutes, discard the supernatant, add 1-2mL of culture medium and blow evenly. Divide the cell suspension into new T25 bottles in a ratio of 1:2, add 6-8ml of new culture medium prepared according to the instructions to maintain the growth vitality of the cells, and proceed with subsequent passages in a ratio of 1:2 to 1:5 based on the actual situation.

Attention: Cell passageIf proliferation can still be maintained after 1-2 cycles, the drug concentration can be increased to 0.5ug/mL for further cultivation; If the cell proliferation stops during this process and the condition is poor, the drug concentration needs to be reduced (by half) or cultured in a medium without drugs until the cell density is about 8 × 105 cells/mL and the growth condition is good, then the required drug concentration should be replaced.

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.

belowTaking T25 bottle as an example;

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 × 106~1 × 107 live cells/ml

Centrifuge at 1000rpm for 3-5 minutes and remove the supernatant. Resuspend the cells in the prepared cell cryopreservation solution, and distribute them into a cryopreservation tube at a concentration of 1 × 106~1 × 107 live cells/ml per 1ml of cryopreservation solution. Label the cells with their names, generations, dates, and other information.

Attention: Do not add drugs during cell cryopreservation.

2. 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.

Human colorectal adenocarcinoma cell line HCT15/DDP resistant to cisplatin