Kinetic imaging of cells

Incucyte S3

The Incucyte S3 is an imaging sytem within an incubator. This enables on-line imaging of cell cultures for time-dependent experiments without disturbing the culture or physical intervention. Images are acquired for up to 6 microtiter plates in all popular SBS-formats. The incucyte features:

  • Fluorescent channel 1 - 480 nm ex /520 nm em
  • Fluorescent channel 2 - 585 nm ex /640 nm em
  • Phase contrast
  • Objectives: 4X/10X/20X

Cell growth assays

Cell motility assays

Migration of a monolayer is performed on cultures plated in image-lock plates and a modification using the 96-well Woundmaker apparatus to create a calibrated region of interest. Plates and apparatus are available in G-INCPM.

Scratch assay

Contact noga.kozer@weizmann.ac.il

New Chemical Libraries

Protein-Protein Interaction Library (Partially supported by the Barry Sherman Medicinal Chemistry Institute)

Protein-protein interactions (PPI) are emerging as tractable targets for small molecules. Although PPIs are challenging targets, some motifs can be targeted for binding inhibitors or stabilizers (aka “molecular glues”). A new library of 40,000 compounds designed by Enamine has been integrated into the G-INCPM screening collection. The library focuses on well-documented protein motifs which have been targeted with small molecules. 20 different protein‐protein complexes highlight specific features of the majority of potent inhibitors in this area. Several specific recognition patterns, like α‐helix, β‐sheet, PDZ‐, PBD and bromodomains were used in the library design. Ligand‐ and Structure based in silico screening, created the selection of compounds featuring:

  • Specific recognition patterns, including hot spots analysis, key amino acids, secondary/tertiary structures α‐helices, ‘hot loops’ and specific protein domains affinity
  • Lead‐like properties and sp3‐rich core structural motifs. Compounds passed all affiliated MedChem filters including compound promiscuity (PAINS)
  • Latest chemistry and novel building blocks. Identified hits can be readily followed with synthesis of new analogs through Enamine REAL Database and with support of the G-INCPM Medicinal Chemistry

Contact haim.barr@weizmann.ac.il

Multiplexed Immunoassays

Luminex

Luminex is a bead-based detection platform for multiplexing up to 50 different simultaneous assays in each well. Luminex or alternatively MAPx (Multi-Analyte Profiling, where the “x” represents biomarkers such as proteins, nucleic acids, or polysaccharides that are being tested). xMAP Technology uses labeled microspheres or beads, allowing for the simultaneous capture of multiple analytes from a single reaction. Because of their small size and low density, xMAP microsphere-based assays exhibit virtual solution-phase kinetics during the reaction. The beads are individually read using MAGpix instrument. Additionally, it is possible to automate sample preparation as well. We can perform the whole process or alternatively you can prepare the plates with the relevant beads and samples and then bring it to read in our center. There are several analytes like for examples cytokines or antibodies against pathogens that are preset up in existing commercial kits. If a commercial kit is not available, we can help you in customizing your beads to enable testing of relevant analytes.

Recent publications from our group using this technology

https://www.jci.org/articles/view/150319

Contact leo.solmesky@weizmann.ac.il for more information

Target-engagement assays

Compound-induced thermal stabilization of proteins is frequently used to confirm binding of small molecule inhibitors to protein targets.

Thermal Shift Assay

Protein stability screenings can be performed using a protein melting method. These can reveal changes in protein structure as a result of their thermal stability. Thermal shift assays (TSA) have traditionally been applied to purified proteins to study their thermal stabilization by the use of dyes with sensitive fluorescence detection (for e.g. SYPRO Orange, Glo-Melt) that enable rapid and relative inexpensive characterization by using a real-time PCR instrument. By screening a wide range of solutions and additives in a 384-well format, we can easily identify conditions that perturb the stability of recombinant proteins. The workflow below (Figure 1) shows our unit’s established methodology for a high-throughput thermal denaturation of a recombinant protein by using a fluorescent dye.

Cell-based thermal shift assay (CETSA)

In recent years, the thermal shift assay armamentaria evolved to include a cellular thermal shift assay (CETSA). Though based on the same principle as conventional TSAs, CETSA allows for ligand induced stabilization in more complex environments like intact cells or cell lysates. This technology is currently offered in our unit for target engagement confirmation following a high throughput screening. This method can currently be applied for small numbers of compounds of high interest and potency.

Recent publications from our group using this technology

https://www.nature.com/articles/s41598-020-77028-8

Contact silvia.carvalho@weizmann.ac.il

Fluorescent-labelled cell panel

Profiling bioactivity of a compound across multiple cell lines it is useful to compare effects in diverse genetic context. We collaborated with the Stem Cell Unit of LSCF to generate an easy-to-use resource of 20 cell lines from different tissue origins, each expressing a stable fluorescent marker. All cell lines have been calibrated with staurosporine as a reference compound. GFP was introduced to cell lines using TALENS according to https://doi.org/10.1002/stem.1653 selected by FACS and propagated in culture.

Recent publications from our group using this technology

https://pubs.acs.org/doi/abs/10.1021/jacs.2c00269

Contact noga.kozer@weizmann.ac.il