Glass slide cell culture

Warning

NBW hasn’t gotten this to work well yet, the DDRR makes MEF move off of them. I would use glass-bottom plates or try a diff coating than gelatin like laminin.

Cleaning Protocol (optional)

If cells are not attaching to glass coverslip try cleaning them. The commercially precleaned ones should not have to be treated like this.

  1. Incubate coverslips overnight in 1N HCl on orbital shaker

  2. Rinse in water at least 3 times

  3. Wrap in foil and autoclave on dry cycle (30 min sterilization time)

Coating glass coverslip

  1. Ethanol glass coverslip sleeve and tweezers, and bring into hood

  2. Carefully place glass coverslip into suitable TC plate, being careful to not touch it with your glove and such that it lays flat

    • It will be difficult to make sure you’re only grabbing a single coverslip

    • A 12 mm round cover glass coverslip should fit into a 24-well plate (16.2 mm ID)

  3. Add enough 0.1% gelatin to coat coverslip, incubate 10 min at RT

  4. Aspirate excess gelatin and seed cells

Mounting glass coverslip

  1. Carefully place a small drop (~10 µL) of mounting solution in the middle of the glass slide where the coverslip will be placed

    • How much mounting solution depends on glass coverslip size, you want enough to cover the whole thing

  2. Carefully use tweezers to remove glass coverslip

    • It will be difficult to grab it without scraping cells off

    • Try to tilt plate one way and lift the coverslip with tweezer

    • Once the slidcoverslipe lifts a bit, carefully push the tweezer a little to shimmy it under the coverslip

    • Softly squeeze the tweezers to grip the coverslip

  3. Place coverslip onto the glass slide such that the cells face down and will be submerged in the mounting solution

    Important

    Avoid bubbles in the mounting solution! It can help to set one edge of the slip into the mounting solution, then slowly set the rest of it down such that the act of putting the coverslip down will push the mounting solution towards the edges.

  4. Let mounting solution set overnight in the dark before moving to a glass slide organizing case

Tip

When mounting, aspirate some of the media off but leave some liquid to help detach the glass slide from the bottom otherwise it will tend to stick to the bottom.