Protocol for Single-Cell Isolation Using Glass Chips

Glass chips have two advantages over other clonal isolation techniques. First, visual observation is generally easier; the presence of a single cell can be easily verified. Second, if you have a steady hand, a fairly large number of chips can be picked in a short period. A disadvantage of the method is that when working with primary cultures or cell lines that have a low cloning efficiency, many chips must be picked before one will be found that will develop into a colony. Another disadvantage is that preparing the chips is tedious.

• Break coverslips into 1 mm² to 2 mm² fragments. (This can be done by a variety of methods. One suggestion is using a mortar and pestle.)
• The fragments are then sifted through two stainless steel soil sieves (1 mm and 0.45 mm diameters). The fragments retained on the 0.45 mm screen are then washed. The larger fragments can be collected and broken into smaller fragments and rescreened.
• Put the coverslip fragments into a large beaker and add equal volumes of water and nitric acid (add acid to water). Then bring to a fuming point (60 to 70°C) on an electric hot plate in a fume hood for 30 minutes. Remove the beaker from the hot plate to cool. When cool, decant the acid into the hood's cup sink with the water running; rinse the chips thoroughly under cold running water for no less than three hours, then rinse six times in distilled water.
• Transfer the sized coverslip fragments to glass tubes with metal closures and dry heat sterilize at 180°C for 3 hours.
• Add 50 to 200 coverslip fragments to each of six 60 mm plastic tissue culture dishes and distribute evenly across bottoms.
• Carefully add 3 ml of growth medium to each plate. Avoid floating the glass fragments.
• Prepare cell suspension by the gentlest means possible.
• Inoculate (using 1 mL of medium) duplicate dishes containing glass fragments with 1, 2, and 5 x 10³ cells per dish. It is important to do a range of cell concentrations. If the initial cell concentration is too high most of the chips will have more than one cell. If it is too low then very few if any chips will have a cell.
• After cells have had sufficient time to attach, (1 to two hours) look for coverslip fragments with a single adherent cell. Start with the cell concentration that has the best ratio of chips with single cells.
• Using watchmaker forceps transfer those fragments with a single cell to separate wells of a 24-well plate containing 1 mL medium/well. Use a conditioned medium if possible; it will increase the cloning efficiency as well as help the cells grow faster. Make sure the cell is on the top side of the glass chip after it is placed into the well or else the cell may be unable to divide.
• Once the surface of the glass fragments is overgrown with cells (usually 5-10 days), it is best to disperse the colony by enzymatic treatment. The cells can then be replicated in a similar culture vessel. When left alone on the glass fragments, colonies will migrate onto the dish surface, but growth is usually slower in this manner.

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• Properly clean and prepare the glass chips before use. This may involve rinsing with ethanol and ultrapure water, followed by drying in a sterile environment. Some protocols may recommend coating the glass chips with specific reagents to enhance cell adhesion or prevent non-specific binding.
• When loading cells onto the glass chip, use precise techniques to ensure uniform distribution and avoid overcrowding. The concentration of the cell suspension should be optimized for single-cell isolation, typically at a low density to increase the chances of isolating individual cells.

Cell Isolation Kit

For research use only. Not for any other purpose.