A549 human lung carcinoma cells

Recommendations for Handling Cryopreserved Cells

1. Receipt and Storage Upon Arrival

Cryopreserved cells are shipped in dry ice. Upon receipt, immediately check that the cryovials remain fully frozen. If any cell thawing is observed in the vial, take photos of the vial before proceeding with any experiments or storage.

If immediate culture is not planned, cryovials must be stored in liquid nitrogen (-196°C) immediately upon arrival; alternatively, they can be temporarily stored at -80°C (note: long-term storage is recommended only in liquid nitrogen).

2. Thawing Procedure

Thaw the cryovial rapidly by agitating it continuously in a 37°C water bath for 45 to 90 seconds. The water bath should be filled with clean water supplemented with an antimicrobial agent. Remove the cryovial from the water bath immediately once the sample is completely thawed (do not over-thaw).

3. Post-Thaw Processing

Transfer the thawed cryovial to a sterile biosafety cabinet and disinfect the outer surface with 70% alcohol. Carefully open the vial and transfer the cell suspension into a 15 ml centrifuge tube containing 9 ml of pre-warmed (37°C) or room-temperature complete cell medium.

To minimize cell damage, add 1 ml of complete cell medium to the empty cryovial, gently pipette up and down to rinse any remaining cells, then transfer this 1 ml suspension to the same 15 ml centrifuge tube. Gently resuspend the cells to ensure uniform mixing, then centrifuge at 300×g for 3 minutes. After centrifugation, carefully discard the supernatant without disturbing the cell pellet.

4. Cell Culture and Passage

Gently resuspend the cell pellet in 5-10 ml of fresh complete cell culture medium. Transfer the resuspended cells into a T25 cell culture flask or a 60-mm diameter cell culture dish.

Follow these steps for subsequent culture and passage:

1. Determine the viable cell count using a suitable method (e.g., trypan blue exclusion assay).
2. Seed the culture dish or flask at a cell density of 1.93-2.94×10? cells/ml.
3. Incubate the cells in a 37°C incubator with a humidified atmosphere containing 5% CO?.
4. Change the medium regularly and perform passage by dilution when the cells reach the appropriate confluency.
5. If the cells are not intended for immediate use, cryopreserve them following standard protocols and store them in liquid nitrogen (-196°C) for long-term preservation.

 

This cell line was established by D. J. Griad through lung cancer tissue transplantation culture. The patient was a 58-year-old white male. A549 can synthesize lecithin containing high content of unsaturated fatty acids through the cytidine diphosphate choline pathway.
cell properties
1)Source: lung cancer
2)Shape: epithelial cell-like, polygonal; adherent growth
3)Content: >1x10^6 cell number
4)Specifications: or Frozen packaging
5)Purpose: For research use only.
transport and storage
Dry ice transport and recovery of viable cells
(1)Frozens are packed with dry ice and transported. After receiving, store in -80 degree refrigerator overnight and then transfer to liquid nitrogen or directly recover. If you find that the dry ice has evaporated completely, the Cryovial caps have fallen off, are damaged, and the cells are contaminated, please contact us immediately.
(2)The recovered viable cells in s are shipped at room temperature. After receipt, follow the handling methods after receiving the cells.
Handling after receipt
1)After receiving the cells, sterilize the bottle wall with 75% alcohol and place the in a 37 C incubator for about 2-3 hours. If you find that the culture bottle is damaged, there is liquid overflow, or the cells are contaminated, please take pictures and contact us in time.
2)Please confirm the cell status under a 4X or 5X microscope. At the same time, take 2-3 photos(10x, 20x)of the newly received cells and a photo of the appearance of the culture bottle to keep as a basis for after-sales service.
3)Adherent cells: Place the cells in a 37 C incubator for 2-3 hours, and observe the growth and adhesion of the cells under a microscope. Some adherent cells will fall off due to vibration during express delivery and form clumps after falling off. If the growth density of the cells is below 60% under a microscope, remove the filling medium from the culture bottle(if there are unadherent cells that need to be recovered by centrifugation, resuspend and put into the original culture bottle), add 6-8 mL of newly prepared complete culture medium, and place it in a cell culture incubator to continue culturing. If the cell growth density reaches above 70%-80%, the cells can be passaged. During the passage process, if cells fall off due to transportation vibration, they need to be recovered by centrifugation.
4)
Note: The transport medium(perfusion medium)cannot be used to culture cells. Please replace it with a new complete medium prepared according to the cell culture conditions in the instructions to culture cells. It is recommended that T25 culture flasks be passaged for the first time after receiving the cells at a ratio of 1: 2.
one. Preparation of culture medium and culture cryopreservation conditions:
1)Prepare F-12K(recommended ahelixbio-0007)medium; high-quality Fetal bovine serum, 10%; double antibody, 1%.
2)Culture conditions: Gas phase: air, 95%; carbon dioxide, 5%. Temperature: 37 C, incubator humidity is 70%-80%.
3)Freezing solution: 90% serum, 10% DMSO, ready for use.
2. Cell processing:
1)Recovery of cryopreserved cells:
Shake the Cryovial containing 1 mL of cell suspension rapidly in a 37°C water bath to thaw, then add it to a Centrifuge tube containing 4-6 mL of complete culture medium and mix evenly. Centrifuge at 1000RPM for 3-5 minutes, discard the supernatant, and resuspend the cells in complete medium. Then add the cell suspension to a culture bottle(or dish)containing 6-8 ml of complete culture medium and culture at 37°C overnight. Observe cell growth and cell density under a microscope the next day.
2)Cell subculture: If the cell density reaches 80%-90%, subculture can be carried out.
For passage of adherent cells, you can refer to the following methods:
1. Discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium and magnesium ions.
2. Add 0. 25%(w/v)Trypsin-0. 53 mM Put EDTA in a culture bottle(1-2 mL for s, 2-3 mL for T75 flasks)and place it in a 37 C incubator for digestion for 1-2 minutes(the digestion time can be appropriately extended for difficult-to-digest cells). Then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take them back to the workbench, tap the culture bottle a few times and add 3-4 ml of culture medium containing 10% FBS to terminate digestion.
3. Beat gently and then aspirate, centrifuge at 1000RPM for 3-5 minutes, discard the supernatant, add 1-2 mL of culture medium and blow evenly. Divide the cell suspension into new s at a ratio of 1: 2, add 6-8 ml of new complete culture medium configured according to the instructions to maintain the growth vitality of the cells, and perform subsequent passages at a ratio of 1: 2 to 1: 5 according to actual conditions.
3)Cell cryopreservation: After receiving the cells, it is recommended to freeze a batch of cell seeds during the first 3 generations of culture for subsequent experiments.
The following is an example;
1. When freezing cells, collect the digested cells into centrifuge tubes according to the cell passage process. You can use a hemocytometer to count the cells to determine the cryopreservation density of the cells. The recommended freezing density for general cells is 1x10^6~1x107 viable cells/ml.
2. Centrifuge at 1000 rpm for 3-5 minutes and remove the supernatant. Resuspend the cells in the prepared cell cryopreservation solution and distribute the cells into a Cryovial according to the concentration of 1x10^6~1x107 viable cells/ml per 1 mL of cryopreservation solution. Distribute the cells into the Cryovial and mark the name, generation number, date and other information.
3. Place the cells to be frozen in a programmed cooling box, keep them in a -80 degree refrigerator overnight, and then transfer them to a liquid nitrogen container for storage. At the same time, record the position of the Cryovial in the liquid nitrogen container for subsequent reference and use.

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