• What cell types can be grown on Lab-Tek Chamber Slide Products, Dishes, Flasks or Coverslips?
• What may be the cause of poor cell growth and attachment?
• What could be the cause of poor resolution experienced when observing cells under the microscope?
• What may be the cause of a cell line not growing well on NUNC Brand products?
• What could be the cause of high incidence of cell death in primary cell culture?
• What reagents are used to coat the Nunclon surface?
• Is the blade on Nunc Cell Scrapers flexible or rigid and from what material is it made?
• Can the Nunc Cell Scrapers be used with the Nunclon TripleFlask?

What cell types can be grown on Lab-Tek Chamber Slide Products, Dishes, Flasks or Coverslips?
Most adherent cell lines and fibroblastic cell types attach to our cell culture treated products. Primary cells can also be cultured successfully on our products with cell culture treated surfaces.

What may be the cause of poor cell growth and attachment?
Many factors which could contribute to poor cell growth and attachment including the type of cell, the age and condition of the culture, culture environment, nutrients, toxins, cell culture protocols, handling and selection of an appropriate surface. The "age" of the cell line or the "passage number" are important parameters to remember when culturing cells. Passage number refers to the number of times the cells have been subcultured. Some cell lines may exhibit morphological changes after several passages. As a cultured population ages, its population doubling time progressively increases until proliferation eventually ceases. In addition, spontaneous cellular transformation may occur with extended time in culture.

Cell culture procedures such as trypsinization can damage cell membranes resulting in poor attachment, clumping or "ragged" looking membranes. Prolonged cultures may result in the buildup of toxins or the depletion of nutrients. Slight changes in culturing conditions (medium, serum, CO₂, temperature, humidity) may result in observed poor growth.

What could be the cause of poor resolution experienced when observing cells under the microscope?
A common problem resulting in poor resolution is a dirty microscope objective caused by fingerprints or oils. The microscope's objective should be cleaned prior to use and always after oil immersion. Also, when using oil immersion, it is important to use the specific brand of oil designed for that particular microscope.

The use of an incorrect type of coverslip or coverglass could also result in poor resolution. No. 1 and No. 1.5 borosilicate coverglass are recommended for the best resolution. The use of plastic coverslips may affect resolution at high magnification due to refractive index differences between the glass and plastic. Also, the thickness of the coverglass is important for clear resolution. For example, No. 2 round coverglass may be too thick for high resolution. Consult your microscope's manual for the best information.

The coverslipping procedure may affect resolution. A clear seal is required, free of debris and particles. When performing oil immersion, bubbles may result in poor resolution under high magnification.

What may be the cause of a cell line not growing well on NUNC Brand products?
Numerous factors influence cell attachment and growth on cell culture vessels. Poor cell attachment can be due to one or more of the following:

• Environmental culture conditions such as temperature, pH osmolality (concentration of various salts in buffer), humidity and the overlying gas phase (CO₂ and O₂ concentration).
• Contamination by microbial agents (bacteria, yeast and molds). Antibiotics are used to minimize the risk of these contaminants. Cell cultures can be contaminated without visible signs of a problem. Viruses and mycoplasma are the most common. Detection of mycoplasma is often difficult. Serum should be obtained from a supplier who certifies it to be free of mycoplasma.
• Components in the medium could be toxic to some cells lines.
• Wash detergent residues left on laboratory glassware may affect cell attachment. Detergent residues may be toxic, in effect solulabilizing the cell membrane or cellular proteins.
• Some cells require an extracellular matrix substrate such as collagen, laminin, fibronectin, polylysine, etc. for optimal growth. Certain cells or cell lines need additional growth factors such as cytokines or nerve growth factors.
• Consult the culture repository where you obtained the cell line for specific information.

What could be the cause of high incidence of cell death in primary cell culture?
Primary cells (cells extracted from tissue) are usually more fragile than established cell lines. This is due to the fact that they have to be extracted (usually by enzymatic digestions) from the surrounding tissues. The choice of digestion enzymes, the length of time these enzymes were used, and how these enzymes were removed is critical to the health and viability of the cells.

What reagents are used to coat the Nunclon surface?
The NUNC Brand Flasks, Plates, Dishes and MultiDishes, Permanox Lab-Tek Chamber Slide Products and Tubes are not coated with any chemical reagents. The Nunclon surface is a surface modification which enhances cell attachment and growth for adherent cell lines.

Is the blade on Nunc Cell Scapers flexible or rigid and from what material is it made?
The Nunc Cell Scraper blade is rigid and it is made from polyethylene. The Nunc Cell Scraper blade is hinged to allow for positioning at different angles for scraping cells using the windshield wiper or hoe method.

Can the Nunc Cell Scrapers be used with the Nunclon TripleFlask?
No, the design of TripleFlask does not allow for mechanical scraping. However, cells are easily harvested using protein digesting enzymes, such as trypsin, collagenase, and pronase or by using non-enzymatic dissociation solutions. Tech Note Vol. 1 No. 2 describes culturing techniques for Nunclon TripleFlask.