Live detection and purification of cells based on the expression of a histone chaperone, HIRA, using a binding peptide

Abstract

Flowcytometry is a reliable method for identification and purification of live cells from a heterogeneous population. Since permeabilized cells cannot be sorted live in a FACS sorter, its application in isolation of functional cells largely depends on antibodies for surface markers. In various fields of biology we find intracellular markers that reveal subpopulations of biological significance. Cell cycle stage specific molecules, metastatic signature molecules, stemness associated proteins etc. are examples of potential markers that could improve the research and therapy enormously. Currently their use is restricted by lack of techniques that allow live detection. Even though a few methods like aptamers, droplet-based microfluidics and smartflares are reported, their application is limited. Here, for the first time we report a simple, cost-effective and efficient method of live sorting of cells based on the expression of an intracellular marker using a fluorophore-tagged binding peptide. The target molecule selected was a histone chaperone, HIRA, the expression of which can predict the fate of differentiating myoblast. Our results confirm that the peptide shows specific interaction with its target; and it can be used to separate cells with differential expression of HIRA. Further, this method offers high purity and viability for the isolated cells.

TM1-IR680 peptide for assessment of surgical margin and lymph node metastasis in murine orthotopic model of oral cancer

“…One of the factors that could affect the specificity of detection is the specificity of the peptide in target detection. We have shown that it interacts only with GRPR by western blot, a modified technique with biotin labelled peptide, which we have published recently24. Further, TM1-IR680-based 3D imaging can detect primary cancer and metastatic lymph nodes, which was not possible by conventional in vivo imaging.…”

ROS‐mediated anticancer effects of EGFR‐targeted nanoceria

“…Since the potential desorption of proteins from nanoparticles happen in the liquid state, it is assumed that the desorption does not occur during the period in which the materials are frozen. FITC-labeled EGF was obtained according to a variant of a process described elsewhere 56 and then conjugated to APTES-nanoceria for confocal imaging studies. EGF was labeled with FITC by mixing FITC and EGF in 1:10 weight ratio in a solution of 0.1 M carbonate buffer at pH 9.0 using a rotary tube mixer at 20 rpm at 4 C under dark conditions for 14-16 h. This was followed by the addition of 1 M NH 4 Cl in order to inactivate the residual FITC.…”