Cell Culture Preparation for Electron Microscopy
Adapted and Prepared by Jeannie Mui
Processing takes three days, with an additional two days for polymerization.
听
Pelleting Cells for EM with 2.5% Glutaraldehyde in 0.1M Sodium Cacodylate Buffer Fixation
1. Cell Collection and Pelleting (suggested)
- Harvest cells from culture by centrifugation:
- Mammalian cells: 500鈥�1,000 脳 g for 5鈥�10 minutes at 4掳C.
- Bacteria: 3,000鈥�8,000 脳 g for 10鈥�15 minutes at 4掳C.
- Carefully remove the supernatant without disturbing the pellet.
2. Washing
- Wash the pellet with ice-cold 1脳 PBS (0.1 碌m sterile filtered) to remove media and secreted proteins.
- Centrifuge again under the same conditions.
- Repeat the wash once more.
3. Primary Fixation
- Transfer the washed pellet to a 1.5 mL Eppendorf test tube filled with听2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight at 4掳C (or up to 72 hours for extended fixation).
- Avoid drying the sample during this step.