Fig 2. Effects of sterol-loading on HeLa cells after cryopreservation.

a) HeLa cells have been enriched with cholesterol by treating them with 0–20 mM methyl-β-cyclodextrin (MβCD) with cholesterol for 60 min. Afterwards their cholesterol content has been measured fluorometrically using enzyme-coupled reactions to produce fluorescent resorufin. n = 3; *: p<0.05 vs 0 mM using student´s t-test b) HeLa cells treated with 0–20 mM MβCD with cholesterol for 60 min have been rapidly frozen without cryoprotectants in open pulled straws (OPS) in liquid nitrogen. After thawing in cell culture medium at 37°C, the plasma membrane integrity of the cells has been tested by propidium iodide (PI) staining (black circles; left y-axis; n = 4–6) and the ability of cells to re-adhere has been tested (blue circles; right y-axis; n = 12). ***: p<0.001 vs 0 mM using student´s t-test c) HeLa cells treated with 0–20 mM unloaded MβCD for 60 min have been rapidly frozen without cryoprotectants in OPS in liquid nitrogen. After thawing in cell culture medium at 37°C, the plasma membrane integrity of the cells has been tested by propidium iodide (PI) staining (black circles; left y-axis; n = 4–6) and the ability of cells to re-adhere has been tested (blue circles; right y-axis; n = 3–5). d) HeLa cells treated with 10 mM MβCD with cholesterol for 60 min (Cholesterol) and untreated control cells have been slowly frozen without cryoprotectants with 1°C/min. After thawing in cell culture medium at 37°C, the plasma membrane integrity of the cells has been tested by PI staining. n = 6; ***: p<0.001 vs control using student´s t-test e) HeLa cells have been enriched with ergosterol by treating them with 0–20 mM ergosterol-loaded MβCD for 60 min. Afterwards their cholesterol content has been measured fluorometrically using enzyme-coupled reactions to produce fluorescent resorufin and their ergosterol content has been measured spectroscopically. Total sterol content is depicted as the sum of the mean values of ergosterol and cholesterol content. n = 3 f) HeLa cells treated with 0–20 mM MβCD with ergosterol for 60 min have been rapidly frozen without cryoprotectants in OPS in liquid nitrogen. After thawing in cell culture medium at 37°C, the plasma membrane integrity of the cells has been tested by propidium iodide (PI) staining (black circles; left y-axis; n = 4–6) and the ability of cells to re-adhere has been tested (blue circles; right y-axis; n = 3–4). *: p<0.05, ***: p<0.001 vs 0 mM using student´s t-test.