Cells were permeabilized as previously described (Adam et al., 1992 (link)). Cells were incubated on ice for 5 min. Then cells were washed in cold transport buffer (TB) (20 mM HEPES, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, and 1 mM ethylene glycol tetraacetic acid (EGTA) at pH 7.3). Cells were then incubated on ice in TB with 34 µg/mL digitonin (Sigma) for 5 min. Cells were then washed twice in cold TB and twice in 37C TB with 1.5% (wt/vol) 360kD polyvinylpyrrolidone (PVP, Sigma). Cells were imaged in 37C TB with 1.5% PVP. In order to confirm that 34 µg/mL digitonin left the nuclear envelope intact but resulted in the permeabilization of the plasma membrane, cells were incubated with 500 ng/mL R-phycoerythrin (ThermoFisher, Waltham, MA) for 10 min and then imaged. R-phycoerythrin is a 240 kD, so it should not readily diffuse through the NPC and can be used as a marker for nuclear integrity.
360kd polyvinylpyrrolidone pvp
Manufactured by Merck Group
360kD polyvinylpyrrolidone (PVP) is a high molecular weight polymer used as a laboratory reagent. It serves as a stabilizing agent and can be used in various applications requiring a water-soluble, non-ionic polymer.
Automatically generated - may contain errors
2 protocols using 360kd polyvinylpyrrolidone pvp
1
Membrane Permeabilization and Nuclear Integrity
2
Nuclear Envelope Permeabilization Assay
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Cells were permeabilized as previously described (Adam et al., 1992) (link). Cells were incubated on ice for 5 min. Then cells were washed in cold transport buffer (TB) (20 mM HEPES, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, and 1 mM ethylene glycol tetraacetic acid (EGTA) at pH 7.3). Cells were then incubated on ice in TB with 34 ug/mL digitonin (Sigma) for 5 minutes. Cells were then washed twice in cold TB and twice in 37C TB with 1.5% (wt/vol) 360kD polyvinylpyrrolidone (PVP, Sigma). Cells were imaged in 37C TB with 1.5% PVP. In order to confirm that 34 µg/mL digitonin left the nuclear envelope intact but resulted in the permeabilization of the plasma membrane, cells were incubated with 500 ng/mL R-phycoerythrin (ThermoFisher, Waltham, MA) for 10 minutes and then imaged. R-phycoerythrin is a 240 kDa, so it should not readily diffuse through the NPC and can be used as a marker for nuclear integrity.
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