Method and kit for assessing viable cells

Abstract

The present invention provides simple, rapid methods and procedures for analyzing cells, hereunder quantitative and qualitative assessment of cells, such as viability. The present invention relates to the use of various optionally substituted reporter compounds particularly detectable upon their reaction with thiol-containing species present in higher concentrations in intact (e.g., living) cells than in non-intact (e.g., dead, stressed and apoptotic) cells. The present invention also relates to the use of various optionally substituted reporter compounds particularly detectable upon their reaction with species present in intact and/or non-intact cells. Moreover, the present invention relates to the use of measuring techniques and/or instruments coupled with the use of various optionally substituted reporter compounds. The invention further relates to compositions used in methods for analyzing cells, such as a composition comprising N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide (DACM).

1 .- 25 . (canceled) 
     
     
         26 . A method for quantitative and/or qualitative assessment of cells, said method comprising:
 providing a sample comprising cells;   adding N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide (DACM) to said sample;   incubating said sample with DACM for less than one hour, whereby said DACM reacts with one or several thiol groups in the cells, producing a labelled sample comprising reacted DACM; and   quantitatively or qualitatively assessing cells in the labelled sample by assessing fluorescence emitted from the reacted DACM.   
     
     
         27 . The method according to  claim 26 , wherein the assessing comprises determination of cell viability. 
     
     
         28 . The method according to  claim 27 , wherein the determination of cell viability comprises assessment of a cell characteristic selected from the group consisting of: metabolic activity, metabolite quantification, cell division, proliferation, health, stress level, apoptosis, necrosis, mobility, spatial orientation and morphology. 
     
     
         29 . The method according to  claim 27 , wherein the determination of cell viability includes quantification of viable cells. 
     
     
         30 . The method according to  claim 26 , wherein the sample is selected from the group consisting of: a body fluid sample, a tissue sample, a fermentation sample, a liquid cultivation sample, a cell culture sample, a water sample, a beverage sample, and a pharmaceutical sample. 
     
     
         31 . The method according to  claim 26 , wherein the sample is a biological sample selected from the group consisting of: a blood sample, a urine sample, a saliva sample, a semen sample, a solubilised tissue sample, and a milk sample. 
     
     
         32 . The method according to  claim 26 , wherein the sample is a biological sample selected from the group consisting of: a liver sample, a kidney sample, a muscle sample, a brain sample, and a lung sample. 
     
     
         33 . The method according to  claim 26 , wherein the sample is a biological sample selected from the group consisting of: a human sample, a mouse sample, a rat sample, a monkey sample, a dog sample, a yeast sample, a bacterial sample, and a protozoan sample. 
     
     
         34 . The method according to  claim 26 , wherein the sample is a biological material selected from the group consisting of: a bacterial culture, a yeast culture, a mammalian cell culture, a protozoa culture, and other cell cultures. 
     
     
         35 . The method according to  claim 26 , wherein the sample is a sample of a material associated with the manufacture, storage, and transportation of a biological material. 
     
     
         36 . The method according to  claim 26 , wherein the labelled sample emits light after excitation by an external light source, producing fluorescence emitted from the reacted DACM. 
     
     
         37 . The method according to  claim 36 , wherein the external light source is focused by a focusing system, comprising one or more lenses, or mirrors. 
     
     
         38 . The method according to  claim 36 , wherein the external light source is selected from the group consisting of: a halogen lamp, or a gas lamp such as a xenon lamp, a light emitted diode (LED), a laser and a laser diode. 
     
     
         39 . The method according to  claim 36 , wherein external light source comprises two or more external light sources. 
     
     
         40 . The method according to  claim 26 , wherein quantitatively or qualitatively assessing cells in the labelled sample comprises producing a magnified image wherein the linear ratio between magnified image and labelled sample is smaller than 10:1 and more preferably smaller than 6:1 and even more preferably smaller than 4:1. 
     
     
         41 . The method according to  claim 26 , further comprising incubating said sample with a dead cell labelling agent. 
     
     
         42 . The method according to  claim 41 , wherein the dead cell labelling agent is propidium iodide (PI). 
     
     
         43 . A method for quantitative or qualitative assessment of biological cells providing a labelled biological sample comprising labelled biological cells to a sample domain, wherein the labelled biological cells are labelled with N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide (DACM), wherein biological cells are incubated with the DACM for less than one hour, whereby the biological cells react with free thiols in said biological sample producing labelled biological cells;
 exposing, onto an array of active detection elements, an at least one-dimensional spatial representation of electromagnetic signals having passed from the domain, the representation being one which is detectable as an intensity by individual active detection elements, under conditions which will permit processing of the intensities detected by the array of detection elements during the exposure in such a manner that representations of electromagnetic signals from the labelled biological cells are identified as distinct from representations of electromagnetic signals from background signals, and wherein the spatial image exposed onto the array of active detection elements is subject to such a linear enlargement that the ratio of the image of a linear dimension on the array of detection elements to the original linear dimension in the exposing domain is smaller than 20:1;   processing the intensity detected by the detection elements in such a manner that signals from the labelled biological cells are identified as distinct from background signals, producing a processing result; and   obtaining a quantitative or qualitative assessment of the labelled biological cells based on the result of the processing.   
     
     
         44 . The method according to  claim 43 , wherein a light source is provided capable of emitting light to the labelled biological cells in the labelled biological sample. 
     
     
         45 . The method according to  claim 44 , wherein the light source is an LED light source. 
     
     
         46 . The method according to  claim 43 , wherein the assessment of the labelled biological cells includes assessment of a characteristic selected from the group consisting of: cell viability, mobility, spatial orientation and morphology. 
     
     
         47 . The method according to  claim 43 , wherein the assessment of cells comprises quantification of viable cells. 
     
     
         48 . The method according to  claim 43 , wherein the assessment of the labelled biological cells comprises assessment of a characteristic selected from the group consisting of: metabolic activity, metabolite quantification, cell division, proliferation, health, stress level, apoptosis, and necrosis. 
     
     
         49 . A method for quantitative or qualitative assessment of apoptotic status of cells providing a sample comprising biological cells;
 adding DACM to said sample, whereby said DACM reacts with one or several thiol groups in the biological cells, producing a labelled sample; and   assessing apoptotic status of biological cells in the labelled sample.   
     
     
         50 . A method of determining the level of free thiols in cells, comprising
 providing a sample with biological cells;   incubating said sample with DACM for less than one hour, whereby said DACM reacts with one or several thiol groups in the cells, producing a labelled sample; and   quantifying cellular fluorescence in the labelled sample, thereby determining a level of free thiols in said biological cells.