6- Introduction to Cell Function Short Course J. Paul Robinson The SVM Professor of Cytomics Professor of Biomedical Engineering Director, Purdue University Cytometry Laboratories @Cytometryman

scintillon.org/nolan_lab Key Links cyto.purdue.edu isac-net.org scintillon.org/nolan_lab Purdue List Serve ISAC Nolan Lab John P. Nolan - jnolan@scintillon.org J. Paul Robinson – wombat@purdue.edu 1:19 PM

Cell Function Flow cytometry can measure many properties that may change over time Cell size may reflect a cellular response Dyes can be added to cells to measure changes such as Ca2+ changes, or pH Fluorescent dyes can measure production or presence of oxygen metabolites such as superoxide anions Toxis effects of drugs may change the number of molecules of various indicators e.g. glutathione 1:19 PM

Flow cytometry can measure function of cells Viability pH Phagocytosis Bacterial killing Effect of toxins Mitochondrial damage Cell metabolism – calcium flux Cell Proliferation Apoptosis (cell death) Membrane polarization 1:19 PM

Why care about viability? It is critical to identify dead cells because dead cells typically take up antibodies which may have fluorescent labels on them – so you must eliminate dead cells This cannot be done in bulk measured systems and even in imaging is more difficult due to the limited number of parameters that can be measured simultaneously There are several dyes that can be used such as PI (Propidium Iodide) 1:19 PM

Propidium Iodide (PI) - Cell Viability How the assay works: PI cannot normally cross the cell membrane If the PI penetrates the cell membrane, it is assumed to be damaged Cells that are brightly fluorescent with the PI are damaged or dead Viable Cell Damaged Cell PI PI PI PI PI PI PI PI PI PI PI PI PI PI 1:19 PM

Cell Function - Oxidative Burst Generation of toxic oxygen species by phagocytic cells Superoxide anion measured with hydroethidine Hydrogen peroxide measured with 2’,7’-dichlorofluorescin diacetate (DCFH-DA) 1:19 PM

Fluorescent Indicators How the assays work: Superoxide: Utilizes hydroethidine the sodium borohydride reduced derivative of EB Hydrogen Peroxide: DCFH-DA is freely permeable and enters the cell where cellular esterases hydrolyze the acetate moieties making a polar structure which remain in the cell. Oxidants (H2O2) oxidize the DCFH to fluorescent DCF Glutathione: In human samples measured using 40 M monobromobimane which combines with GSH by means of glutathione-S-transferase. This reaction occurs within 10 minutes reaction time. Nitric Oxide: DCFH-DA can indicate for nitric oxide in a similar manner to H2O2 so care must be used. DAF is a specific probe available for Nitric Oxide 1:19 PM

? ? Human Neutrophil Stimulant (PMA) PKC Phospolipase A2 activity Leukotrienes Lipid Peroxidation OH. Phagosome H2O2 O2- + H2O + O2 H+ ? Stimulant H2O2 O2 SOD Catalase (PMA) O2- PKC O2- SOD ? GSSG H2O2 NADPH GP Oxidase GR GSH PCB NADPH + H+ H2O NADP+ HMP PCB PCB 1:19 PM (Reduced GSH level)

Neutrophil Oxidative Burst PMA-Stimulated Neutrophils Scale Log DCF Unstimulated Neutrophils 345 115 38 12 4 600 1200 1800 2400 TIME (seconds) 1:19 PM

DCFH-DA DCFH DCF Fluorescent Hydrolysis Oxidation 1:19 PM H2O2 DCFH-DA 2’,7’-dichlorofluorescin diacetate COOH H Cl O O-C-CH3 CH3-C-O 2’,7’-dichlorofluorescin COOH H Cl OH HO O Fluorescent Cellular Esterases 2’,7’-dichlorofluorescein Hydrolysis COOH H Cl O HO H2O2 Oxidation DCFH-DA DCFH DCF H O 2 2 Lymphocytes Monocytes Neutrophils log FITC Fluorescence .1 1000 100 10 1 20 40 60 counts PMA-stimulated PMN Control 80 1:19 PM

HE EB Hydroethidine O2- O2- H2O2 Example: Neutrophil Oxidative Burst CH2CH3 NH2 H2N H Br- N CH2CH3 NH2 H2N + Phagocytic Vacuole NADPH Oxidase NADPH O2 HE O2- NADP DCF SOD O2- H2O2 DCF H2O2 OH- Example: Neutrophil Oxidative Burst 1:19 PM

Hydroethidine - Superoxide Production Endothelial cells also respond to Oxygen indicators 15 minutes 45 minutes Carter, W. O., Narayanan, P. K., Robinson, J. P: .Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells Journal of Leukocyte Biology 55: 253-258, 1994

Phagocytosis Uptake of Fluorescent labeled particles Determination of intracellular or extracellular state of particles How the assay works: Particles or cells are labeled with a fluorescent probe The cells and particles are mixed so phagocytosis takes place The cells are mixed with a fluorescent absorber to remove fluorescence from membrane bound particles The remaining fluorescence represents internal particles 1:19 PM FITC-Labeled Bacteria

Organelle Function Mitochondria Rhodamine 123 Endosomes Ceramides Golgi BODIPY-Ceramide Endoplasmic Reticulum DiOC6(3) Carbocyanine 1:19 PM

Oxidative Reactions Superoxide Hydroethidine Hydrogen Peroxide Dichlorofluorescein Glutathione levels Monobromobimane Nitric Oxide Dichlorofluorescein 1:19 PM

Superoxide measured with hydroethidine cell 1 Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis. cell 2 cell 3 cell 4 cell 5 Step 6C: Export data from measured regions to Microsoft Excel 1800 1600 cell 1 cell 2 cell 3 cell 4 cell 5 1400 base to Delta Graph Step 7C: Export data from Excel data 1200 1000 %change (DCF fluorescence) 800 600 400 200 -200 200 400 600 800 1000 1200 1400 1600 1800 Time in seconds 1:19 PM

Calcium Flux Flow Cytometry Image Cytometry Time (seconds) 1:19 PM 0.8 0.7 0.6 0.5 Ratio: intensity of 460nm / 405nm signals 0.4 0.3 0.2 Stimulation 0.1 36 72 108 144 180 Time (seconds) Time (Seconds) 50 100 150 200 1:19 PM

Membrane Potential Oxonol Probes Cyanine Probes How the assay works: Carbocyanine dyes released into the surrounding media as cells depolarize Because flow cytometers measure the internal cell fluorescence, the kinetic changes can be recorded as the re-distribution occurs PMA Added fMLP Added Repolarized Cells Green Fluorescence 0 512 1024 0 512 1024 Green Fluorescence Depolarized Cells 0 1200 2400 150 300 Time (sec) Time (sec) 1:19 PM

Membrane Polarization Polarization/fluidity Diphenylhexatriene How the assay works: The DPH partitions into liphophilic portions of the cell and is excited by a polarized UV light source. Polarized emissions are collected and changes can be observed kinetically as cells are activated. Membrane polarization can be measured by both imaging and flow cytometry An image showing DPH fluorescence in cultured endothelial cells. 1:19 PM

CD16 Expression on Normal Cultured PMN Function can be measured by expression of receptors. CD16 is a low affinity Fc receptor. It is a cluster of differentiation molecule found on the surface of natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes and macrophages. negative control 24 Hours As cells age, the CD16 expression reduces. The “bright” CD16 antigen is lost first 0 Hours 48 Hours 1:19 PM

Ionic Flux Determinations Calcium Indo-1 Intracellular pH BCECF How the assay works: Fluorescent probes such as Indo-1 are able to bind to calcium in a ratiometric manner The emission wavelength decreases as the probe binds available calcium Time (Seconds) 36 72 108 144 180 Stimulation 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 50 100 150 200 Ratio: intensity of 460nm / 405nm signals Time (seconds) Flow Cytometry Image Analysis 1:19 PM

Light Scatter Changes of PMN at 24 Hours control Damaged/highly activated 1:19 PM

Cell Proliferation Data from Center for AIDS Research UCDS 1:19 PM http://cfar.ucsd.edu/core-facilities/flow-cytometry/services-and-pricing-and-instruments/flow-cytometry-services 1:19 PM

Cell Function Summary Flow cytometry can measure many parameters that can detect minute changes to cells This can be cell size or structure/complexity Receptor expression may change as a function of activation Phagocytosis or pH can be measured on a cell by cell basis Metabolite production is easily measured 1:19 PM