The aim of cryopreservation is to enable stocks of cells to be stored to prevent the need to have all cell lines in culture at all times. It is invaluable when dealing with cells of limited life span. The other main advantages of cryopreservation are:

• Reduced risk of microbial contamination
• Reduced risk of cross contamination with other cell lines
• Reduced risk of genetic drift and morphological changes
• Work conducted using cells at a consistent passage number
• Reduced costs (consumables and staff time)

There has been a large amount of developmental work undertaken to ensure successful cryopreservation and resuscitation of a wide variety of cell lines of different cell types. The basic principle of successful cryopreservation is a slow freeze and quick thaw. Although the precise requirement may vary with different cell lines as a general guide cells should be cooled at a rate of –1oC to –3oC per minute and thawed quickly by incubation in a 37oC waterbath for 3-5 minutes. If this and the additional points given below are followed then most cell lines should be cryopreserved successfully.

1. Cultures should be healthy with a viability of >90% and no signs of microbial contamination.
2. Cultures should be in log phase of growth (this can be achieved by using pre-confluent cultures i.e. cultures that are below their maximum cell density and by changing the culture medium 24 hours before freezing).
3. A high concentration of serum/protein (>20%) should be used. In many cases serum is used at 90%.
4. Use a cryoprotectant such as Cell Culture Plate(6 well plate, 24 well plate, 96 well plate) or glycerol to help protect the cells from rupture by the formation of ice crystals. The most commonly used cryoprotectant is DMSO at a final concentration of 10%, however, this is not appropriate for all cell lines e.g. HL60 where DMSO is used to induce differentiation. In such cases an alternative such as ELISA Plate should be used (refer to data sheet for details of the correct cryoprotectant). Sigma also offers ready-made cell freezing media containing DMSO , glycerol and a serum-free formulation containing DMSO.

Source: SIGMA-ALDRICH

 How are glass bottom cell culture dishes typically used?

1. Maintain sterility: Open dishes in a sterile environment (e.g. laminar flow hood).

2. Pre-equilibrate dishes: Incubate the dishes with culture medium. Pipet 2-3 ml of medium into the 35 mm glass bottom cell culture dishes or 3-4 ml into the 50 mm glass bottom cell culture dishes and incubate at 37° C for 15 minutes.

3. Add cell suspension to microwell: Remove the culture medium by aspiration and plate cells onto the glass surface. Pipet 250μl of the cell suspension (cells suspended in culture medium) into the 10 mm diameter microwells or 500μl of cell suspension into the 14 mm microwells. Incubate the dishes for 1 hour at 37° C.

4. Add additional medium: After 1 hour, gently fill the remainder of the dish with medium Add 2-3 ml to the 35 mm glass bottom cell culture dishes or 3-4 ml for the 50 mm glass bottom cell culture dishes. Note: After the initial one hour period to allow cells to attach to the glass surface, it is important to fill the dish to normal levels in order to minimize the effects of evaporation and to avoid inducing changes in osmolarity.

Biousing 35mm Glass Bottom Cell Culture Dishes Product Features:

Glass bottom dishes have a thin and optically clear bottom. A clinically non-toxic adhesive is used to attach the cover glass to the bottom of the TC treated culture dish. Round cover glass is used to make this product more elaborate.

 A Microtiter plate (spelt Microtitre in Europe) or microplate is a flat plate with multiple “wells” used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories. A very common usage is in the enzyme-linked immunosorbent assay (ELISA), the basis of most modern medical diagnostic testing in humans and animals. A microplate typically has 6, 24, 96, 384 or even 1536 sample wells arranged in a 2:3 rectangular matrix. — from Wikipedia

Biousing Biotechnology Co., Ltd. is a professional biology hi-tech company that is focused on research, manufacturing and sales of high-level laboratory consumables, devices and reagents. Their featured ELISA produt is 96 well ELISA plate.

Features of 96 well ELISA plate:

Produced conform to the SBS-3d-standard exhibit finest workmanship. The binding capacity is 400-500ng/ cm2, CV<4%.

The bottom thickness of biousing 96 well cell culture plates are carefully lessened. This change assures less edge effect because the temperature will reach uniform faster.

The absolutely flat floor area, free from inclusions, guarantees the highest level of transparency. The detection background is lower than that of the other international brands.

The smoothness of Biousing cell culture plates is much better than the best-selling brands. This will greatly eliminate the system error when 96-well plates are used in ELISA.

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