Nuclear Apoptosis Assay Cell Line
A SH-SY5Y/TagRFP-LaminB1 cell line and a U2OS/TagRFP-LaminB1 cell lines has been developed through stable transfection for monitoring the cellular apoptosis level through nuclear lamin morphological changes in cell-based assays
Each vial of LMNB1 Apoptosis Assay Cell Line contains more than 3 million cells stably expressing human lamin B1 (LMNB1) tagged in the N-terminus with TagRFP.
Assay Details:To measure the apoptosis levels, cell with TagRFP-LaminB1 were stimulated with staurosporine. In non-apoptotic cells TagRFP appears localized in nuclear envelope but after treatment, activated caspase family proteases cleave lamin, resulting in the loss of its structure and detachment from chromatine. Tagged lamin appears disassembled and forms round aggregates.
The assay was developed and optimized using the BD Pathway HCS Reader and Attovision Compartimentalization Software. The parameters analyzed in order to check the apoptosis degree are eccentricity and perimeter of the nucleus.
The parameters to be analyze in order to check the apoptosis degree are the nuclear eccentricity and the nuclear perimeter. This assay has been validated based on the first parameter with an average of Z´=0.72 +/- 0.01 for High Content Screening.
Fig 1: TagRFP-LaminB1 without (A) and with 10 μM (B)staurosporine.
Fig 2: Nuclear eccentricity assay curve. In this curve, the Ec50 for staurosporine was 0.32 μM aftera treatment of 6h. Activity was calculated relative tocontrol. This assay was validated with an average of Z’= 0.72+/- 0.01 for High Content Screening.
Fig 3: Nuclear perimeter assay curve. In this curve, the Ec50 for staurosporine was 0.13 μM after a treatment of 6h. Activity was calculated relative to control. This assay was validated with an average of Z’= 0.54+/- 0.01 for High Content Screening.