Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
  • G01N33/5735—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes co-enzymes or co-factors, e.g. NAD, ATP
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
  • G01N2333/80—Cytochromes

Definitions

• the present invention refers to a method to quantify mitochondrial cytochrome c release during cellular death comprising selective permeabilization of the plasma membrane and cytochrome c immunodetection.
• Apoproteic cytochrome c is a component of the mitochondrial respiratory chain found on the external side of the inner mitochondrial membrane, which is released from the mitochondria to the cytosol after the triggering of the apoptosis process in various kinds of cell in response to various cell aggressions. Once the cytochrome c is released, along with cytosolic factors, it activates a cascade of proteases, termed caspases, which promote cell death. Given the fact that the mitochondrial release of cytochrome c is a critic step in the apoptosis process, its quantification may be used to characterize this type of cellular death.
• the known methods to measure the release of cytochrome c comprise Western blot and fluorescence microscopy.
• the Western blot technique requires the fractioning and separation of mitochondria from homogenized cell. After this stage the fractions undergo long and laborious procedures, including electrophoresis of the proteins through a gel and from this gel to a membrane, membrane incubation with primary and secondary antibodies to detect cytochrome c . Finally the cytochrome c is developed and quantified to indicate the degree of cell death. This procedure usually requires at least two days. Furthermore, during the cell homogenization, the mitochondria may break and the Western blot technique could lead to a super estimation of the cytochrome c release.
• Fluorescence microscopy is both faster and easier than the mentioned technique, but the analysis of apoptotic cells under the microscope may also be long and, more relevant, the sampling is always the main concern whenever this technique is used.
• the method described according to the present invention remarkably reduces the amount of time spent in establishing cytochrome c mitochondrial release, from at least two days to one day, while it enhances the precision on measuring and the number of analyzed cells during apoptosis.
• it is possible to perform the analysis using any method among fluorescence, chemo luminescence, calorimetric and cytometric .
• Our method is based on the differences between the lipids that make up the plasma membrane and the mitochondrial membrane. The cholesterol/phospholipid ratio is very high on the plasma membrane, whereas either the mitochondrial inner and outer membrane ratios are low because they have very little or even no cholesterol at all .
• digitonin cholesterol-specific detergents
• the present invention comprises a method to quantify the mitochondrial cytochrome c release during cell death comprising the following steps: a) selective permeabilization of the plasma membrane; b) cytochrome c immunodetection.
• the step of selective permeabilization of the plasma membrane precedes the cytochrome c immunodetection and analysis by fluorescence, chemo luminescence and chromatometry techniques .
• the invention comprises a composition to detect mitochondrial cytochrome c release, which include a primary antibody against cytochrome c, a secondary antibody associated to fluorochrome, digitonin and buffers.
• the invention comprises, still, a kit for detection of the released mitochondrial cytochrome c including primary antibody against cytochrome c, secondary antibody associated to fluorochrome, digitonine and buffers.
• the invention comprises, also, the use of a composition to quantify the released mitochondrial cytochrome c during cell death including primary antibody against cytochrome c, secondary antibody associated to fluorochrome, digitonin and buffers .
• HL-60 leukemic cells treated with Staurosporine, a protein kinase C inhibitor were used as model for apoptosis induction. These cells undergo apoptosis, quantified by the external!zation of phosphatidylserine via anexin V-FITC, when maintained in culture medium for 18 hours in the presence of the drug. Apoptosis is observed during the first 6 hours of exposure to the drug, although apoptotic signals have already been triggered inside the cells.
• Figures 1 to 9 illustrate the fluorescence histograms of cells labeled with antibody against cytochrome c analyzed by flow cytometry.
• the histograms illustrate the time-dependent decrease of HL-60 cells fluorescence exposed to Staurosporine as a consequence of cytochrome c release.
• the cells treated with Staurosporine for 1 to 6 hours and permeabilized with digitonin presented progressively lower fluorescence levels up to near the background fluorescence, as indicated by the fluorescence average (FM) , inside each histogram ( Figures 4 to 9) , suggesting that after 6 hours all cytochrome c has been released from the mitochondria.
• Experimental control exhibit untreated cells permeabilized or not with digitonin ( Figure 4 and Figure 3, respectively) . Both presented similar levels of high fluorescence, indicating that the low digitonin concentrations used did not release a significant amount of cytochrome c from the mitochondria.
• unlabeled cells Figure 1 or cells labeled only with secondary antibodies ( Figure 2) presented fluorescence on background levels .
• the HL-60 cells were incubated, or not, with 1 ⁇ M Staurosporine for 6 hours in a RPMI medium supplemented by 10% FBS, 2 mM L-glutamine, in an atmosphere of 5% C0 2 at 37°C.
• 10 6 cells were collected and washed twice with phosphate buffered saline solution (PBS) and centrifuged at 1000 g during 5 min.
• PBS phosphate buffered saline solution
• the cells were once again suspended in a 100 ⁇ l PBS solution supplemented with a mix of protease inhibitors at 1% plus 1 mM PMSF.
• the cells were vigorously shacked for 30 seconds with 0.005% digitonine.
• the volume was rectified with PBS to 10 ml, centrifuged at 5000 g for 5 min and the pellets fixated with a 2% paraformaldehyde for 20 min. After two washings in PBS under identical conditions, the cells were washed in labeling medium containing PBS, 2% fetal calf serum, 0.5% Triton X-100, 0.2% sodium azide and centrifuged at
• Figure 1 presents the result of the background fluorescence for untreated HL-60 cells.
• Figure 2 is the background fluorescence for the secondary antibody (2° Ab) of untreated cells.
• Figure 3 is the maximum fluorescence for untreated cells not permeabilized and incubated with primary (1° Ab) and secondary antibody.
• Figure 4 exhibits the maximum fluorescence of untreated cells after the permeabilization of the cells with digitonin and incubation with both antibodies.
• Figures 5 to 9 illustrate the reduction of fluorescence on HL-60 cells treated with Staurosporine incubated with both antibodies .
• the average fluorescence means the average value of the fluorescence in the region where the fluorescence is highest.

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Cited By (2)

• 2003
  • 2003-02-28 BR BRPI0300660A patent/BRPI0300660B8/pt not_active IP Right Cessation
• 2004
  • 2004-02-26 WO PCT/BR2004/000022 patent/WO2004077007A2/en active Application Filing

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