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Cell culture

Cell culture

First, project introduction

Cells are the basic structural and functional units of organisms. It is known that all organisms except viruses are composed of cells. Therefore, in-depth study of cells is the key to uncovering the mysteries of life, transforming lives, and conquering diseases. Taking cells as the research object, it is of great significance to study the structure and function of cells and organelles, the life history of cells, and the laws of various life activities from three levels: the overall level of cells, the submicroscopic level, and the molecular level.

Second, sample storage and transportation (when sending cells, please download the sample delivery information sheet "cell sample delivery form" from the data center and fill in and send it to the platform assistant)

1. Live cell transportation method

Please use an air-tight culture flask as shown in the figure to culture cells. When the cell confluence reaches about 70%, fill with fresh medium to close to the bottle mouth, tighten the bottle cap, and seal the bottle mouth with the sealing film. Mark the bottle (cell name, medium, date, etc.). Shockproof treatment must be done when packing. Sent at room temperature.

2, The method of transporting frozen cells

Be sure to ensure sufficient dry ice (2-3 days delivery time, 8-10 kg dry ice is sufficient), and the cryotube must be completely covered by dry ice. Do not freeze and thaw. Do not mix water ice into dry ice pieces.

3. Precautions for receiving streaming samples

① Apoptosis

requires live cells (no samples from other places). If the cells are fluorescent, be sure to make a note on the transfer list and culture plate, otherwise treat as non-fluorescence. If the test results are unavailable and samples are wasted as a result, we will not be responsible and will not accept refunds.

②Cell cycle

The  cycle requires a large amount of cells, and the 6-well plate must have a confluence of more than 70%, but not more than 90%. Samples from the field need to be fixed overnight with pre-cooled 70% ethanol (adherent cells need to be digested and fixed), centrifuge to discard the fixative, add 1ml of PBS to resuspend, seal with a plastic film, and ship to the laboratory at 4°C.

③Flow JC-1

requires live cells (no samples from other places). The cell confluence is higher than 70% (6-well plate), and 2 more blank holes are provided for negative and positive controls. If the cell has red or green light, it cannot be detected.

④Flow ROS

requires live cells (no samples from other places). The cell confluence is higher than 70% (6-well plate), and 2 more blank holes are provided for negative and positive controls. If the cell is green, it cannot be detected.

⑤Cell surface resistance

Determine in advance whether the company has the corresponding flow antibody (histochemical antibody is not available), other antibodies need to be provided by the customer (no samples from other places).

⑥Streaming triple standard

Inform the specific number and type of samples (blood or spleen~) in advance. The blood sample is collected with an anticoagulant tube (at least 1.5 mL for intracellular staining, and about 1 ml for epitope). Take care to prevent coagulation and store and transport at 4°C. Blood samples can only be stored at 4°C for one week at most. The longer the time, the worse the results will be. After the blood is taken, it must be sent to the laboratory as soon as possible; when taking the spleen, try to bring the animal to the laboratory, and start the experiment directly after taking the material from the laboratory. If the animal cannot be taken to the laboratory, take the material (the entire spleen) and place the tissue in Store in an EP tube or centrifuge tube containing PBS at 4°C. Cell extraction must be completed within 6 hours after sampling. Note that some test indicators (Treg, TH1, TH2, etc.) need to be extracted aseptically. Samples from other places will not be accepted.

4, Plasmids, RNAs, viruses

Plasmid vector, miRNA, siRNA, etc. are sent at -20°C.

Lentivirus: Store at -80℃, and send it with plenty of pure dry ice.

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