Single-cell analysis of apoptosis associated molecules

                              Vojtěch Ledvina^1,2, Karel Klepárník^1

    ^1Institute of Analytical Chemistry of the Czech Academy of Sciences, Brno, Czech Republic

                  ^2Faculty of Science, Masaryk University, Brno, Czech Republic


Caspases are proteases that are traditionally associated with apoptosis, the programmed cell death,
but novel non-apoptotic functions of caspases were reported as well, especially as modulators of
cell differentiation and proliferation. Recently, caspases were reported as molecules partaking on
differentiation of mouse osteoblastic cells. Analyzing cell lysates from large sets of cells often
obscure the cell-to-cell variations within the cell culture or tissue. It is therefore desirable to
analyze the caspases activation at the single-cell level. To address these needs, we have developed
a miniaturized device for the single-cell analysis of caspase activation that utilizes commercially
available bioluminescence chemistry based on modified aminoluciferin and firefly luciferase. The
developed technique was subsequently used for analysis of caspase activation during MC3T3-E1
osteoblastic cell line differentiation.

Fas ligand (FasL) is a transmembrane protein that is also closely associated with apoptosis as it
is the initiator of the extrinsic apoptotic pathway. Its polarization on the surface of cells can
play a key role in tissue remodeling for example during bone formation. In our work, we have
developed functionalized cathodoluminescent nanoparticles based on lanthanide doped LuAG and
NaYF[4]that were conjugated with antibodies against the FasL. The nanoparticles provide both
fluorescent and cathodoluminescent signal and they have potential for application in correlative
light and scanning electron microscopy.

The research was supported by the Grant Agency of the Czech Republic, project no. 17-01995s (at the
IAC ASCR, v.v.i.) and by the Institute of Analytical Chemistry of the CAS under the Institutional
Research Plan RVO: 68081715.