Direct attachment of cell suspensions to high-pressure freezing specimen planchettes.
We describe a procedure for high-pressure freezing (HPF) of cultured cells using the HPF aluminium planchettes as a substrate. Cells are either grown directly on planchettes covered with Matrigel or allowed to attach to poly-l-lysine-coated planchettes. This method allows for rapid transfer of the cells into the HPF and minimizes physical and physiological trauma to the cells. Furthermore, the yield of well-frozen cells approaches 100% for every cell type we have tried so far. In this report, we show well-preserved ultrastructure in mitotic and interphase HeLa cells, isolated gastric parietal cells and isolated gastric glands. Immunogold labelling of H+/K+-ATPase is shown in parietal cells of isolated gastric glands embedded in LR White resin. The aluminium planchettes appear to have little effect on cell physiology, as demonstrated by the fact that parietal cells cultured for 24-28 h on the planchettes retain their responsiveness to stimulation with histamine.
D000535:Aluminum; D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D015925:Cryopreservation / Q000295:instrumentation* / Q000379:methods*; D017110:Freeze Substitution; D005753:Gastric Mucosa / Q000166:cytology / Q000648:ultrastructure; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008854:Microscopy, Electron; D010295:Parietal Cells, Gastric / Q000648:ultrastructure; D011312:Pressure; D011817:Rabbits
D000535:Aluminum; D017506:H(+)-K(+)-Exchanging ATPase; D008239:Lysine