Use of tumor dissociation reagent in flow cytometry

Abstract

The present disclosure relates to a dissociation reagent for tumor tissues. The dissociation reagent does not contain collagenase or trypsin but further contains hyaluronidase or a mixture of hyaluronidase and DNase I. The present disclosure also relates to use of the dissociation reagent in dispersing tumor tissues and detecting expression level of molecular markers on cell surface by flow cytometry. The dissociation reagent of the present disclosure does not cause degradation of molecular markers on cell surface such as CD8, PD-1, Tim-3, Lag-3 and the like, thus does not affect downstream assays.

1 . A tumor dissociation reagent which does not comprise collagenase but comprises hyaluronidase, wherein the tumor dissociation reagent does not degrade or partially degrade membrane surface receptor. 
     
     
         2 . The tumor dissociation reagent of  claim 1 , which further does not comprise trypsin. 
     
     
         3 . The tumor dissociation reagent of  claim 1 , which further comprises DNase I. 
     
     
         4 . The tumor dissociation reagent of  claim 1 , wherein the membrane surface receptor is a checkpoint receptor. 
     
     
         5 . The tumor dissociation reagent of  claim 1 , wherein the membrane surface receptor comprises CD8, PD-1, PD-L1, TIM-3 or LAG-3 protein. 
     
     
         6 . A method for detecting protein expression level of an immunological checkpoint marker in tumor tissue, comprising applying the tumor dissociation reagent of  claim 1  to the tumor tissue. 
     
     
         7 . The method of  claim 6 , wherein the tumor dissociation reagent further comprises DNase I. 
     
     
         8 . The method of  claim 6 , wherein the tumor tissue includes tumor infiltrating immune cell. 
     
     
         9 . The method of  claim 6 , wherein the protein expression in tumor tissue is detected by flow cytometry. 
     
     
         10 . The method of  claim 6 , wherein the protein is membrane surface receptor. 
     
     
         11 . The method of  claim 10 , wherein the membrane surface receptor comprises CD8, PD-1, PD-L1, TIM-3 or LAG-3 protein. 
     
     
         12 . A kit for tumor dissociation comprising the tumor dissociation reagent of  claim 1 . 
     
     
         13 . The kit of  claim 12 , wherein the tumor dissociation reagent further comprises DNase I. 
     
     
         14 . A method of detecting protein expression in tumor tissue by flow cytometry comprising applying the kit of  claim 12  to the tumor tissue. 
     
     
         15 . A method of preventing degradation of an immunological checkpoint marker in tumor tissue, comprising treating cells with the tumor dissociation agent of  claim 1 . 
     
     
         16 . The method of  claim 15 , wherein the tumor dissociation reagent further comprises DNase I. 
     
     
         17 . The method of  claim 15 , comprising dissolving the tumor tissue with the tumor dissociation reagent, and detecting the immunological checkpoint marker by flow cytometry. 
     
     
         18 . The method of  claim 17 , wherein the tumor tissue includes tumor infiltrating immune cell.