Cell Culture Preparation for Morphological Electron Microscopy
Adapted and Prepared by Jeannie Mui
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.
Preparing Cell Monolayers for Electron Microscopy Processing
-
Cell Culture
- Seed and culture cells in an 8-well Nunc™ Lab-Tek™ Chamber Slide System equipped with Permanox™ slides.
- Maintain cells under appropriate growth conditions until they reach approximately 80-90% confluency. Avoid overgrowth, as excessive confluency may affect cell morphology and ultrastructural preservation.
-
PBS Washes
- Carefully aspirate the culture medium from each well.
- Gently wash the cell monolayers twice with pre-warmed phosphate-buffered saline (PBS) to remove residual culture medium, serum proteins, and cellular debris.
- Minimize the duration of the washes to prevent cellular stress or detachment.
-
Primary Fixation
- Add sufficient 2.5% glutaraldehyde prepared in 0.1 M sodium cacodylate buffer at room temperature to completely cover the cell monolayer.
- Ensure all wells are fully immersed in fixative and free of air bubbles.
- Transfer the chamber slides to 4°C and allow fixation to proceed overnight (12-18 hours) to achieve optimal preservation of cellular ultrastructure.
-
Sample Transport
- Following fixation, keep samples refrigerated and protected from temperature fluctuations.
- Transport the chamber slides to the FEMR using cold packs to maintain a cool temperature during transit.
- Do not ship or transport samples on dry ice, as freezing may damage the fixed cell monolayer and compromise sample quality.
-
Additional Notes
- Clearly label all samples with the investigator's name, sample identification, cell type, and date of fixation.
- Notify FEMR staff before sample submission if special processing requirements or experimental conditions need to be documented.
- Handle fixed samples with care to avoid disruption or detachment of the cell monolayer.