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Proteins and peptidesAnti-Ly6g antibody [1A8] - mouse IgG2c (Chimeric)
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Our first-to-market chimera with mouse IgG2c backbone, this functional antibody specifically depletes neutrophils in vivo for up to 72h.
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Cells require careful maintenance to prevent contamination, facilitate growth and ensure long-term stability. The following pages provide general guidance for preserving, thawing and maintaining
Show moreCells in culture can broadly be classified into the following three types.
Types of cell culture | Meaning |
Adherent | Cells adhere to the culture vessel (e.g. tissue culture plastic). |
Suspension | All cells grow in suspension with the growth medium, and do not attach to the culture vessel to grow. |
Semi-adherent | Some cells adhere loosely to the culture vessel and others may remain in suspension in the growth medium. |
Cells can also be classified according to their growth characteristics, and bring their own set of considerations for growth:
Here our focus will be on the most common cell types; adherent and suspension cells. Please note: all procedures involving the manipulation of cultured cells should take place using aseptic technique and the appropriate containment method(s).
Genetic instability accumulates in cells that are continually cultured. Therefore, cell lines should be frozen and stored, or “banked down”, as soon as possible after receipt. This ensures that cell stocks are as genetically as close as possible to the source material and reduces risk of contamination. Cells should be frozen in a controlled manner (ideally at a rate of 1°C per minute) in the presence of cryoprotective agents (such as DMSO) to prevent the formation of ice crystals within the cells and a resulting loss in viability of the culture.
Note: our cell lines arrive frozen in cryoprotectant and should be immediately stored in liquid Nitrogen upon receipt.
Ensure your culture is healthy and in a logarithmic phase of growth
Use a microscope visualise the cells to check their general appearance and ensure there are no signs of microbial contamination.
The cell density should not exceed the guidance range for your cell line and viability should be > 90 %, although this depends on the cell line.
Collect and count the cells as described for standard sub-culture
Suspension cells are usually cryopreserved at a density of 2 – 5 x 106 cells per mL, Adherent cells at 1 – 2 x 106 per mL
Wash and prepare cells
Resuspend cells in cryoprotectant according to manufacturer’s instructions
Pros and cons of different cryoprotectants
Types of cryoprotectant | Pros | Cons |
DMSO (usually 5 – 10% in serum or serum- containing media) | Inexpensive | DMSO can adversely affect some cell types. Need to prepare the solution yourself; may be less consistent than pre-prepared. |
Glycerol (usually 2 – 20% in serum) | Inexpensive Some evidence that viability may be higher upon thawing than with DMSO. Non-toxic | Less effective than DMSO for prevention of ice crystal formation and osmotic shock. Need to prepare the solution yourself; may be less consistent than pre-prepared. |
Pre-prepared solutions (eg Bambanker™) | Consistency in formulation
| Expensive |
The choice of cryoprotectant will depend on a number of factors including the sensitivity of the cells being frozen.
Freeze cryovials
Pros and cons of different freezing containers
Type of freezing container | Pros | Cons |
Isopropyl alcohol based (eg Mr Frosty™) | Improvement upon “handmade” freezing containers | Requires manual addition of alcohol to container and monitoring |
Alcohol-free polyethylene (e.g. CoolCell®) | Improved standardization over alcohol-based containers | Higher cost |
Cells should be frozen in specially designed freezing containers. These are designed to ensure a controlled rate of -1 to -3 °C per minute to minimize cell damage during the freezing process
Transfer to liquid Nitrogen (vapor phase) for long-term storage
When required for use, cells should be thawed as quickly as possible to minimize any adverse impact on cell viability. It is recommended that the cryopreservation agent is removed from the culture medium by centrifugation at time of revival.
Thaw cryovial in a water bath
Specific seeding densities, centrifuge speeds, and incubation conditions can be obtained from the relevant cell line repository.
Remove cryopreservation agent and seed cells for culture
Cells should be observed regularly using a microscope and with the unaided eye for signs of microbiological contamination. Microscopic examination should also be used to determine the general health of the cells and to establish whether subculture is required.
Observe cell culture by naked eye for visible markers of growth or contamination
Temperature levels, CO2 and metabolism of ingredients can affect the pH of growth medium.
Observe cells under a light microscope for signs of growth or contamination
Cells should continually be monitored for signs of bacterial, fungal and yeast contamination. It is also possible for different cell lines to contaminate cultures.
Based on your observations, implement the appropriate course of action.
Remove growth medium
Add fresh growth medium
Incubate cells as required
Continue to monitor cells daily for growth and signs of contamination