Cell Culture Preparation for Electron Microscopy

Adapted and Prepared by Jeannie Mui

Processing takes three days, with an additional two days for polymerization.
 

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: 500–1,000 × g for 5–10 minutes at 4°C.
  • Bacteria: 3,000–8,000 × g for 10–15 minutes at 4°C.
• Carefully remove the supernatant without disturbing the pellet.

2. Washing

• Wash the pellet with ice-cold 1× PBS (0.1 µm 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 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight at 4°C (or up to 72 hours for extended fixation).
• Avoid drying the sample during this step.