Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell divisionCell division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

Potential Applications of Flow Cytometry Calcium fluxCalcium flux Levels of intracellular reactive oxygen speciesLevels of intracellular reactive oxygen species Identification of “stem cells”Identification of “stem cells” Telomere lengthTelomere length......

Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell divisionCell division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

Activation IL-7 67% Medium 23% Activation FSC x SSC – Cell size

Activation CD69, CD71, etc

Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell divisionCell division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

Cell Cycle Analysis DNA content analysis - Propidium Iodide

Cell Cycle Analysis DNA content analysis - Propidium Iodide

Cell Cycle Analysis DNA content analysis - Propidium Iodide Careful with doublets! DNA Content G0/G1 S Phase G2/M Or doublets of G0/G1 cells?

Cell Cycle Analysis DNA content analysis - Propidium Iodide Careful with doublets!

Cell Cycle Analysis DNA content analysis - Propidium Iodide G 0 -G 1 S G 2 -M Fluorescence Intensity Cell Number

Cell Cycle Analysis Propidium Iodide plus BrdU staining Thymidine analog Taken up by cells in S-phase Usually in combination with Propidium iodide Cell cycle analysis - Bromodeoxyuridine (BrdU) method New Click-It DNA technology from Invitrogen does not require DNA denaturation.

Cell Cycle Analysis Propidium Iodide plus BrdU staining Propidium Iodide Anti-BrdU FITC G1 G2/M S Phase

Cell Cycle Analysis Pyronin Y plus Hoechst 33342/33258 – G0-G1 discrimination

Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell divisionCell division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

Tracking cell divisions with CFSE CFSE: Carboxy-fluorescein diacetate, succinimidyl ester (fluorescein molecule containing a succinimidyl ester functional group and two acetate moieties) Diffuses freely into cells Intracellular esterases cleave the acetate groups converting CFSE into a fluorescent, membrane impermeant dye. Not transferred to adjacent cells. Retained by the cell in the cytoplasm Does not adversely affect cellular function During each round of cell division, the relative intensity of the dye is decreased by half.

STAIN WITH CFSE DILUTION OF CFSE Cell Divisions

IL-7 IL-7+ DMSO IL-7+ PI3K Inhibitor IL-7+ Erk Inhibitor

Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell DivisionCell Division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

CELL DEATH – FSC x SSC

Apoptosis Propidium Iodide (fixed cells)

Apoptosis Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells)

Apoptosis Annexin V plus propidium Iodide

Apoptosis Annexin V plus propidium Iodide

(intracellular staining) Fix and permeabilize Add Antibody Analyse by Flow Cytometry Apoptosis Bcl-2 family members

Apoptosis Bcl-2 family members Bcl-2

Apoptosis Bcl-2 family members Flow cytometric analysis of Raji cells, using Bim Antibody (IF Preferred) (blue) compared to a nonspecific negative control antibody (red). Bim But …

Apoptosis Activated forms of caspases Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate). Untreated Etoposide

Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell DivisionCell Division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

Analysis of T cell development CD4-CD8-CD3- (TN) CD4+CD8-CD3- (ISP) CD4+CD8+CD3-/lo (DP) CD4+CD8+CD3+ (TP) CD4+ CD8-CD3+ (SP4) CD4- CD8+CD3+ (SP8)

Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell DivisionCell Division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

Cytokine Secretion (intracellular cytokine staining) Activate T cell Add inhibitor of protein export (ex: Brefeldin A; Golgi-STOP) Fix and permeabilize Add Cytokine- Specific Antibody Analyse by Flow Cytometry

Cytokine Secretion (intracellular cytokine staining) anti-hIL-2 Ab

Stimulate + Brefeldin A Incubate Wash and fix Stain for surface antigens Wash Permeabilize Wash Stain with cytokine- specific antibodies Intracellular Cytokine Staining Plus Surface Staining

Intracellular Cytokine Staining Plus Surface Staining IFN-  CD8 / CD69

Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell DivisionCell Division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

Activation of signalling pathways (phospho-protein detection)

Activation of signalling pathways

Discrimination of High vs. Low responders P-STAT6

Activation of signalling pathways

Discrimination of simultaneous vs. non-simultaneous activation of different pathways in single cells

Combining surface markers with Phospho-staining

PFA/saponin protocol failed to detect P-STAT5 (but...better for surface staining and better cell recovery) Activation of signalling pathways IMPORTANCE OF FIXATION METHOD

Analyse by Flow Cytometry Telomere length by Flow FISH Tetramer-CY5 (and/or Ab) + Crosslink to cell surface Denature +Probe Blood 2001 vol 97, 700 Hybridise Permeabilise

CD45RA Telomere CD4 + CD45RA + gate CD4 + CD45RA - gate Telomere Telomere Length in naïve and memory CD4+ T cells

MULTIPLEXED BEAD ARRAYS Can use arrays of beads to measure multiple cytokines at once on the one sample Can use arrays of beads to measure multiple cytokines at once on the one sample Each type of bead coated with capture antibody for particular cytokine Each type of bead coated with capture antibody for particular cytokine Mix beads with sample, wash, then add anti- cytokine ab with fluorchrome reporter Mix beads with sample, wash, then add anti- cytokine ab with fluorchrome reporter Amount of fluorescence measured proportional to amount of cytokine bound to each bead (equivalent to ELISA OD reading) Amount of fluorescence measured proportional to amount of cytokine bound to each bead (equivalent to ELISA OD reading)

NEAT1/8 Y-axis FL3 discriminates 5 bead types X-axis is reporter fluorescence for amount of cytokine bound to bead 1/64NEG

Y-axis FL3 discriminates 5 bead types X-axis is reporter fluorescence for amount of cytokine bound to bead MOUSE 1 MOUSE 2

Future Advances More colours for immunofluorescence (quantum dots, tandem dyes) More colours for immunofluorescence (quantum dots, tandem dyes) Reduced laser size and capillary flow techniques mean smaller instruments Reduced laser size and capillary flow techniques mean smaller instruments Instruments can now image cell at point of laser interrogation Instruments can now image cell at point of laser interrogation

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