Instruments available in the NIBN and related applications

Analyzingsorting cells Available parameters: 1. Size vs. density 2. Protein expression 3. DNA content

Common Application • • Detection, characterization and sorting of rare sub populations Cell cycle analysis Cell Vitality Pluidity level assays in plants Enrichment of libraries in directed evolution experiments Analysis of stained nanoparticles Analysis of bacterial cells expressing GFP Enrichment of a GFP/BFP expressing cell population after transfection • Detecting unhomologous recombination events in Arabidopsis cells via levels of GFP or GUS

FACS instruments NIBN SY 3200 Sorter (Sony) Eclipse EC 800 (Sony) SP 6800 Spectral Analyzer (Sony) Joint NIBNHealth science department Galius (Beckman Culter) Health science department Canto (Becto Dickenson)

Fluorescence Activated Cell Sorting 1. Cells are labeled with one or more fluorescent markers

Fluorescence Activated Cell Sorting 2. Labeled Cells are exposed (one by one) in a liquid stream onto a laser beam 525/50 561 DLP 640 DLP 700/75 488 nm PMT 585/40 CD 4 PMT 585/40 PMT 525/50 CD 25 3. Emitted light is collected by a set of PMTs after passing various filters and is transformed onto an electronic signal

Fluorescence Activated Cell Sorting 2. Cells can be sorted according to their fluorescent properties PMT G 1 585/40 525/50 488 nm 640 DLP + 525/50 CD 25 G 1 - 700/75 585/40 CD 4 561 DLP PMT

SY 3200 A high speed Fluorescence Activated Cell Sorter 2 independent sorter modules each equipped with 3 -4 lasers HAPS 1 HAPS 2 375 nm 405 nm 488 nm 561 nm 640 nm 594 nm SY 3200 installed in a Bio safety cabinet (BSC) Colors: FSC, SSC and 6 fluorescent channels Location: Cytometry unit, room 309, building 39

Available filters 792/50 825 LP 455/50 490/20 525/50 577/25 585/40 595/50 605/70 605 LP 615/30 665/30 700/75 736 LP 775/50 780/60

SY 3200 A high speed Fluorescence Activated Cell Sorter Can sort up to 20, 000 cells/sec into 4 sub populations High sensitivity Can separates 0. 2 um, 0. 3 um and 0. 5 um particles Auto-mix agitation station for long sort procedures on each module Single cell deposition unit for sorting single cells into 384/96 well plates A BSL 2 approved sterile environment

Eclipse cell Analyzer (EC 800) Lasers: 488, 640 Colors: FSC, SSC, 5 fluorescent channels, EV channel Location: Cytometry unit room 309 building 39.

Optical Configuration Lasers: 488 n. M 642 n. M

Auto sample loader FACS tubes Eppendorf 1. 5 ml Tubes Eppendorf 0. 5 ml tubes 24 -384 wells plate Flat/ V shaped/ U shaped bottom Standard/ deep well configuration Eight reagent holder positions are available for on board sample preparation including programmable volume, suspension, and incubation options.

Canto II (Becto Dickenson) Lasers: 405, 488, 640 Colors: FSC, SSC and 6 channels Autoloader for Plates Location: Health science department

Gallios (Beckman Culter) Lasers: 405, 488 and 561, 637 Colors: 10 channels Autoloader for FACS tubes Location: Health science department

Excitation and emission curves

SP 6800 Spectral analyzer Std. analyzers 530/30 SP 6800 585/42

SP 6800 Spectral analyzer

Key benefits of spectral analysis

Affinity measurements Protein – protein interactions Protein – small molecule interactions Protein – DNARNA interactions

Common application • Measuring DNA-Protein, Protein-protein and protein- small molecules kinetics • Screening Antibodies with different peptides • Testing enzyme – inhibitor interactions • Examining mutations effect on protein-protein interactions • Effect of sugar modifications on protein-protein binding

Affinity measurements NIBN Surface Plasmon Resonance (SPR) - Prote. On XPR 36 TM Micro. Scale Thermophoresis (MST) - Monolith

Prote. On XPR 36 TM Measures Kinetics and affinity between molecules A label free system A wide variety of chips for binding of His-tagged, biotin conjugated or unmodified ligands A unique 6 x 6 conformation enables the measurement of 36 interactions at the same run Location: Cytometry unit, room 309 building 39

Surface plasmon resonance An SPR experiment Step 1 Bind up to 6 ligands Activate • Immobilize • Deactivate • Step 2 Inject up to 6 analytes perpendicularly Step 3 Detail showing One of 36 interaction spots

One shot kinetic experiment: 5 ligands tested vs 6 analyte concentrations

Surface plasmon resonance Available chips: GLC Carboxyl groups GLM Carboxyl groups GLH Carboxyl groups 5000 -8000 RU 8000 -13000 RU up to 21000 RU NLC Neutravidin Biotin tagged Molecules HTG tris-NTA His tagged proteins

Monolith Micro. Scale Thermophoresis Measures Affinity between molecules • • • Small amounts of materials needed No need for binding onto a chip Fast experiment time (10 to 30 minutes) Can work in cell lysate and blood serum If using a GFP/YFP conjugated protein, no labeling is needed Location: Cytometry unit, room 309 building 39

Micro. Scale Thermophoresis Detecting the interaction between two molecules: 1. Label a ligand of interest with a small fluorescent molecule 2. Mix the labeled ligand with various dilutions of the binding partner in capillaries 3. Use MST technology – temporarily heat one point of the capillary to create a thermal gradient and monitor the labeled molecule’s movement 4. Deduce the affinity by observing the effect of complex creation on its movement in thermal gradient

Micro. Scale Thermophoresis Aptamer–thrombin binding in 10% human serum

Prote. On Vs. Monolith Prote. On 1. A High throughput instrument 2. The 6 x 6 configuration enables one to: Measure affinity of a single analyte vs several ligands on a single run Run multiple analytes on the same set of ligands 3. A label free system MST 1. Only small amounts of materials are needed 2. No need for binding onto a chip 3. Fast experiment time (10 to 30 minutes) 4. Can work in cell lysate and blood serum 5. If using a GFP/YFP conjugated protein, no labeling is needed 6. Fast operational learning curve 7. Non aggressive labeling minimizes interference to binding

Cell imaging • Light microscope for cell cultures • EVOS® FLoid® Cell Imaging Station • Holomonitor M 4 for live cell imaging • Scanning Confocal Microscope (Olympus) • Operetta (Perkin Elmer) • Pannoramic automated digital slide scanner

EVOS® FLoid® Cell Imaging Station Fast and easy acquisition of up to 3 color fluorescent images Location: building 41, one instrument in each floor

Holomonitor M 4 for live cell imaging Cell Morphology (Glioma) Monitoring cell morphology and motility inside the incubator Free label system Location: Room 210, building 41 Tracking cell movement

Applications: • • Label free toxicity studies Cell Morphology changes Cell motility tracking Cell division Cell count Macrophage polarisation Chemoresistance studies Check out http: //www. phiab. se/applications

Scanning confocal microscope 4 lasers: 405, 488, 543, 640 3 fluorescent channels High resolution lens (X 60, 1. 35 NA) Pending upgrade: • Replacing the 543 nm laser with a 561 nm laser. • Adding a spectral imaging module • Adding a Silicon oil lens (X 40, 1. 25) Location: Cytometry unit, room 309 building 39

Scanning Confocal Microscope (Inverted) slide 1. Single plane scanning 2. 3 D picture construction 3. Co-localization 4. Improved resolution Concentrating lens Fluorescence from the focal plane only Laser source

Actin (green) and Microtubuli (red) labeling in a Drosophila bristle cell (cross sections) Courtesy of Anna Melkov (Uri Abdu’s lab)

Colocalization of PSD 96 and Shaker. B in Schneider cells Courtesy of Nitzan Zandany and Shir Marciano

Confocal microscope improved resolution Kidney cells Fluorescent microscope Confocal microscope

Operetta A high throughput fluorescentconfocal scanning microscope 1. 2. 3. 4. 5. Fast scanning of samples on a slide or on a multi well plate Eight filter excitation wheel for maximum coverage of excitation dyes A 14 bit CCD camera for high detection sensitivity Environmental control unit for live cell imaging Self learning algorithm for fast and easy data analysis Location: Cytometry unit, room 309 building 39

High throughput scanning microscopy Operetta: Excitation filters/Dyes

Setting up an experiment Tubulin (green) and Nuclear staining (Hoechst 33342) in He. La cells (courtesy of PE labs)

High throughput scanning microscopy Operetta data analysis Step 1: use segmentation to define wanted area of analysis

High throughput scanning microscopy Operetta data analysis Step 2: Calculate various parameters in each segmented area

High throughput scanning microscopy Operetta data analysis Step 3: Select sub populations by applying various filters

High throughput scanning microscopy Operetta data analysis Step 4: display your results

High throughput scanning microscopy Operetta • • • Lipid Droplet Analysis Apoptosis • Neurite Outgrowth Cell Cycle • Protein Expression Cell Differentiation • Receptor Activation Cell Migration • RNAi Screening Cell Proliferation • Signaling Pathway Analysis Cell Shape Changes Cytoskeletal Rearrangement • Transcription Factor Cytotoxicity Fluorescence in situ hybridization (FISH) Ready made solutions (RMS’s) are available

Panoramic Automated Digital slide scanner 12 slides auto loading tray Brightfield, DAPI/FITC/TRITC/Cy 5 filters Advanced acquisition and analysis software Location: Room 002 building 39

Two Photon Microscope The advantages of using two photon confocal microscopy: Better imaging of live tissue (penetration >1 mm) Two-photon microscopy image of a cerebellar lobule. Alanna Watt/ Michael Häusse Better z section resolution Superior signal-to-noise resolution Reduced phototoxicity. Location: Israel Sekler, Health science department

GelECL doc systems Fusion FX for Fluorescence and chemiluminescence documentation Minibis Gel doc. system RGB filters ECL Quantitative analysis software UV and visible light table Location: Room 221 building 41 Location: building 41, in each floor

M 7™ Compact MRI Dedicated Mouse and Rat MRI 3 D whole body morphological imaging (up to 700 g) Applications in: • • Cancer Research Cardiovascular Contrast-based Molecular Imaging Diabetes and Obesity Embryology / Developmental Biology Nephrology Neurobiology Stem Cell / Cell Tracking Location: Animal house, Health science department

Histology unit Contains the following instruments: 1. 2. 3. 4. 5. An automated microtome Tissue fixation instrument Tissue processor A water bath and a flattening table A flattening table- Slide drier. Needs training before use! Location: Room -118 building 39

Guidelines and contact info Interested in using one of these instruments? Be careful! Asking your unexperienced lab mate to teach may result in a you making a big mistake. Contact Dr. Alon Zilka azilha@bgu. ac. il 08 -6428451 Room 309 building 309 I will teach you how to properly use and fully exploit each instrument Or, refer you to an expert user for training. Check out the unit link for more information on these instruments: http: //lifeserv. bgu. ac. il/wb/azilha/