Recombinant differential anchorage probes that tower over the spatial dimension of intracellular signals for high content screening and analysis.
Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.
Schembri
Laura
L
INSERM U916, Institut Bergonié, Université Victor Segalen, 229 cours de l'Argonne, 33076 Bordeaux, France.
Zanese
Marion
M
Depierre-Plinet
Gaelle
G
Petit
Muriel
M
Elkaoukabi-Chaibi
Assia
A
Tauzin
Loic
L
Florean
Cristina
C
Lartigue
Lydia
L
Medina
Chantal
C
Rey
Christophe
C
Belloc
Francis
F
Reiffers
Josy
J
Ichas
François
F
De Giorgi
Francesca
F
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002453:Cell Cycle; D016923:Cell Death; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D049109:Cell Proliferation; D055808:Drug Discovery; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D057054:Molecular Imaging; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D010447:Peptide Hydrolases; D053148:Caspase 3; D053179:Caspase 7