FixationofCellsCulturedinTranswellDishes

Fixation of Cells Cultured in Transwell Dishes

Transwell culture dishes are commonly used to culture cells so that the top and bottom of the cells can be exposed to different culture media conditions. They are commonly used with polarized epithelial cells and endothelial cells. The cells are cultured on membrane supports and processing for transmission electron microscopy is greatly facilitated if the membranes are left intact and under the tension placed by the wells in the dish. Most dishes are destroyed by solvents used in embedding but the following method, when used with polycarbonate Transwell plates obtained from Costar, works very well.

After the embedding resin is hardened, cut the wells with a saw and trim and mount for thin sectioning.

• Solutions:

• 200mM NaCacodylate, pH 7.4

  2.5% glutaraldehyde in 100 mM NaCacodylate

  500 mM CaCl₂

  2% OsO₄ + 1% freshly prepared potassium ferricyanide

  Ethanol for dehydration

• Procedure:

• 1. Remove the culture medium using a pipette.

  2. Fix 30 min with glutaraldehyde in 100 mM NaCacodylate. To improve membrane preservation, add 0.5 mM CaCl₂.

  3. Rinse 3 X with 100 mM cacodylate buffer.

  4. Fix 1 hr, room temp, with 2% OsO₄, 100 mM cacodylate buffer + 1% freshly prepared potassium ferricyanide to increase membrane contrast.

  5. Rinse 3X with 100 mM cacodylate buffer

  6. Dehydrate through 25, 50, 75, 95, and 2X 100% ethanol, 10 min/change. Note: One can leave samples in 75% ethanol at 4 degrees C or 100% resin at room temp overnight.

  7. Infiltrate resin as follows (do not add DMP-30 until the final change )
     

     Resin mix:	Epon 55g
     	DDSA 35g
     	NMA 21g/td>

     
     Mix well and store without DMP-30 at -20 degrees C.
     

     Just add enough resin to cover the cells in the transwell and the bottom side of the wells.

  • 2 hrs 33% plastic, 67% ethanol (100%)

  • 2 hrs 66% plastic, 34% ethanol (100%)

  • 3 hrs, 100% resin

  • 3 hrs, 100% resin with 0.1 ml DMP-30/5 ml resin

  8. Cure at 65 degrees C for 2-3 days