Methods, compositions and kits for assaying mitochondrial function

Patent metadata

Field	Value
Publication number	US-2016282337-A1
Application number	US-201615087521-A
Country	US
Kind code	A1
Filing date	Mar 31, 2016
Priority date	Apr 8, 2011
Publication date	Sep 29, 2016
Grant date	—

Abstract

The invention provides methods, compositions, devices, and kits relating to the use of cholesterol-dependent cytolysins (e.g., PFOs) for measuring intracellular mitochondrial activity.

First claim

1 - 104 . (canceled)

105 . A method comprising:
(a) incubating a cell with a cholesterol-dependent cytolysin, thereby selectively permeabilizing the plasma membrane of the cell; and (b) introducing an exogenous agent into the permeabilized cell.

106 . The method of claim 105 , wherein the exogenous agent is a nucleic acid.

107 . The method of claim 105 , wherein the exogenous agent is protein.

108 . The method of claim 105 , wherein the exogenous agent is an antibody.

109 . The method of claim 105 , wherein the exogenous agent is a dye or fluorescent protein.

110 . The method of claim 105 , wherein the dye is a calcium sensitive dye or a pH-sensing dye.

111 . The method of claim 105 , wherein the exogenous agent is a reducing agent, a respiratory chain inhibitor, an oxidative phosphorylation inhibitor, an uncoupling agent, a transport inhibitor, an ioophore or a Krebs cycle inhibitor.

112 . The method of claim 105 , wherein the exogenous agent is a test agent.

113 . The method of claim 105 , further comprising measuring the effect of the exogenous agent on the cell.

114 . The method of claim 113 , wherein said measuring comprises measuring an intracellular function of the cell.

115 . The method of claim 114 , wherein the intracellular function is a mitrochondrial function.

116 . The method of claim 105 , wherein the cell is a mammalian cell.

117 . The method of claim 105 , wherein the exogenous agent cannot cross the plasma membrane of a non-permeabililzed cell.

118 . The method of claim 105 , wherein the cholesterol-dependent cytolysin comprises an amino acid sequence selected from Table 1.

119 . The method of claim 105 , wherein the cholesterol-dependent cytolysin is Perfringolysin O (PFO) or a variant thereof.

Assignees

• Baystate Medical Center Inc
• Univ Massachusetts

Inventors

• Yadava Nagendra
• Heuck Alejandro Pablo
• Kim Chul

Classifications

• G01N33/5005

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title

• G01N33/5079 Primary

  Mitochondria · CPC title

• C07K14/33 Primary

  from Clostridium (G) · CPC title

• G01N2333/33

  from Clostridium (G) · CPC title