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IP isolates a specific antigen from a solution using a corresponding Ab

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Identification and quantification of proteins from a lysate

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Culture iPSCs in a non-adherent dish to promote the formation of spheroid clusters

iPSC candidates not allowed to stick down so they should behave like ES cells and aggregate to form an embryoid body (like an embryo but disordered) that is dividing in the centre and differentiating on the outside

Confirm by microscopic observation

Also aids in differentiation of iPSCs

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Follow co-immunoprecipitation with mass spectrometry to identify and quantify interacting proteins

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Inject iPSC candidates into 'nude' (immunodeficient) mice to test whether they form teratomas with a mixture of cell types from the three germ layers

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Stained slices through a CiPSC-induced teratoma

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Teratoma formation assay ensures that iPSCs candidates can differentiate into products of all three germ layers

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1. Labeling of iPSC Candidates: To visually track the integration and contribution of iPSCs in developmental processes

Utilize Green Fluorescent Protein (GFP) to label iPSC candidates --> allows for real-time visualization and subsequent analysis of cell integration and behavior in the developmental context

2. Microinjection into Blastocysts: Carefully microinject the GFP-labeled iPSCs into a host blastocyst

3. After microinjection, the blastocysts should be cultured under conditions that support embryonic development

4. Tracing and Confirmation: Confirm that the GFP-labeled iPSCs integrate into the host tissue and contribute to various cell types across the developing embryo; pluripotency of iPSCs is confirmed if they can contribute to tissues in all three germ layers

Utilize fluorescence microscopy to observe the distribution and intensity of GFP expression across different tissues and developmental stages

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1. Lyse cells in detergent --> SDS PAGE against MWM to seperate proteins

2. Separated proteins are transferred out of the gel to the surface of a membrane

3. Membrane is exposed to Ab specific to the target protein , excess Ab washed off

4. Binding of Ab is detected using radioactive or chemical tag or coupled to an enzyme that catalysed a chromogenic or luminescent reaction e.g. alkaline phosphatase (could be via a secondary Ab that binds the primary Ab) --> intensity of band proportional to the amount of target protein

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Analysis of the physical and chemical characteristics of cells as they flow in a fluid stream through a laser beam

Light scattered is characteristic to the cells and their components

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Examine and determine the expression of intracellular molecules and the cell surface and to define and characterize distinct single cell type

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Cells, proteins DNA etc can be labelled with fluorescent markers

If a fluorescent marker is bound to a cellular component, the fluorescence intensity will ideally represent the amount of that particular flow cell component

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Cell counting

Cell sorting

Determining cell characteristics

Biomarker detection

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Can measure the expression and activation of specific signalling molecules on a large number of cells quickly --> provides data of heterogeneity within populations of cells

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DNA binding dyes (PI or DAPI) --> determine percentage of cells in different phases of the cell cycl

DNA in the cells bind the dye stoichiometrically

Flow cytometric analysis of cell count versus linear fluorescence is used to create a histogram of the DNA content distribution across the steps of the cell cycle

G0/G1: DNA content = 2N

S: 2N < DNA content 2N < 4N

G2M: DNA content = 4N

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FACS physically sorts a heterogeneous mixture of cells into different populations

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FACS physically sorts a heterogeneous mixture of cells into different populations

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Operator predetermines or selects certain parameters on how cells should be sorted

Cell sorting machine imposes an electrical charge on each cell so that cells will be sorted by charge (using electromagnets) into separate vessels upon exiting the flow chamber

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How to Complete Cell Cycle Analysis via Flow Cytometry

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How to Complete Cell Cycle Analysis via Flow Cytometry

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How to Complete Cell Cycle Analysis via Flow Cytometry

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Check out our latest blog for a helpful guide on how to complete cell cycle analysis via flow cytometry. Read on to learn more now.

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Co-IP captures protein complexes --> determine interaction between a POI and other proteins

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Confocal microscopy: Imaging technique to obtain high resolution images of cells --> visualise and trafficking of signalling molecules within cells, provides spatial context to signalling events

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Screening for iPSCs by visual inspection: Should appear similar to ESCs

High nucleus-to-cytoplasm ratio (less heterochromatin --> less compact)

Divide and self-renew indefinitely

Colonies should be spherical and well-defined with smooth edges

Stick together due to specific E-cadherin expression (don't 'like' to contact other cells)

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e.g. Test for pluripotency markers on the protein level: Validate the presence of pluripotency cell surface proteins TRA-1-60, TRA-1-81, SSEA-3, and SSEA-4 through immunostaining (and can follow with flow cytometry)

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Immunocytochemistry for pluripotency genes was performed on the GFP+ colonies

Spheres indicated that one cell has divided frequently to produce many daughter cells --> iPSC candidate

Robust experiments test for multiple pluripotency markers in different ways at different levels (RNA, protein) for higher confidence and minimisation of artefacts

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Study the dynamics of protein interactions and movements in real-time in living cells

Popular technique for investigating molecular transport events such as diffusion, protein dynamics, binding dynamics, or interactions between cellular components

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Specific area of a cell or tissue is photobleached by intense laser light, removing fluorescence from this area

Fluorescence in the bleached area will slowly recover as bleached fluorophores move out and healthy fluorophores from other areas move in

Speed of fluorescence recovery of the bleached area is directly proportional to the diffusion speed of fluorescent or labeled molecules --> estimate diffusion coefficient of the labelled molecule

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By bleaching a component or organelle within a cell (such as the nucleus), you can then observe the rate of diffusion of molecules in and out of that component as the fluorescence recovers

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Sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adosrption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA)

After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen

The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation

Between each step, the plate is typically washed with a mild detergent olution to remove any proteins or antibodies that are non-specifically bound

After the final wash step, the plate is developed by adding an enzymatic substrateto produce a visible signal, which indicates the quantity of antigen in the sample

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PNPP (p-Nitrophenyl Phosphate, Disodium Salt) turns yellow when detecting alkaline phosphatase

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Selectively silence the expression of components of signalling pathways --> deduce function from knockdown phenotype

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Genetically encoded biosensors that fluoresce in response to changing conditions (e.g. calcium level, cAMP level, enzyme activity) to monitor real time signalling events within cells

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Fluorescent protein based biosensors change their fluorescence properties in response to specific cellular events

Calcium sensor GCaMP derived from a fusion of GFP, calmodulin and M13 peptide fluoresces brightly when Ca2+ bind calmodulin, indicating increased intracellular Ca2_

cAMP sensor Epac-based sensor changes FRET properties in response to cAMP levels

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Detects PPIs and PTMs

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Two Abs targeted to two proteins or a protein and a modification (e.g. Ub, pTyr, Sumo etc.)

Abs have attached oligonucleotides

If the two ABs are in close proximity, the oligonucleotides ligate --> rolling circle replication --> detectable signal --> easy readout of PPIs and PTMs

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Lineage tracing with Cre-Lox recombination

In cells with no Cre recombinase activity, GFP under a ubiquitous promoter is not expressed due to a LoxP-flanked premature STOP codon

Cre activity stimulates recombination between LoxP sites to remove STOP codon --> GFP expressed

LoxP recombination is permanent --> GFP expressed in all descendants of the cell that had Cre activity

Cre expression can be driven by stem cell-specific promoter if known (e.g. PAX7 promoter for muscle, Lgr5 for intestinal stem cells) --> allows tracing of the stem cell lineage, can identify all the cell types the stem cell produces

OR ubiquitous promoter driving CreERT2 expression: CreERT2 is a chimeric Cre linked to oestrogen receptor, only active in the presence of the oestrogen analog tamoxifen, which can be added in dilute amounts to randomly activate Cre and thus cause GFP expression in a few cells --> non-stem cell linages will eventually disappear and no longer express GFP, stem cell lineages will persist --> allows identification of stem cells

Cre expression can also be driven by tissue/developmental stage-specific promoter --> allows knocking out or over-expressing of a particular gene product at a particular stage (space and time) in development

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RT-PCR was used to show CiPSCs had expression profiles of pluripotency genes similar to ESCs and OKSM-iPSCs

Gapdh used as a loading control (necessary to show equal loading!!)

Variation reflects the fact that, although all pluripotent, there is some variation at the genome level between iPSCs

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Microarrays consist of a small, solid support (usually a glass slide) onto which DNA molecules are fixed in a grid-like pattern, each spot on the grid represents a different gene.

These arrays are used to measure the expression levels of large numbers of genes simultaneously

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Gene expression profiling for the identification of iPSCs: Should express many of the key pluripotency genes characteristic of ESCs e.g. OCT4, SOX2, KLF4, c-MYC, NANOG and REX1 can be used as pluripotency markers

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Microarray data can be represented in a heat map

The gene expression data obtained from a microarray experiment can be displayed in a heat map to visualize differences in expression across various samples or conditions

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A heat map graphically represents the transcriptomic analysis data for visualizing the expression levels of thousands of genes across multiple samples simultaneously, allowing researchers to identify patterns and hierarchical clustering, revealing relationships and similarities between the samples

Red for high expression, green for low expression, and black for medium or unchanged expression

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Bisulphite genomic sequencing showed that CiPSCs had low levels of DNA methylation at the Nanog and Oct4 promoters (indicating 'active' genes) similar to ESCs and in contrast to differentiated MEFs

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Oct4-GFP observed in mice testis confirms germline transmission possible to create a line of transgenic animals

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Nanog-GFP expression can be used as a marker for stem-ness in the initial screen

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Epigenetic assessment for identification of iPSCs: Test that promoters and histones of pluripotency genes are de-methylated and epigenetically-primed for transcription using meDIP-seq, bisulphite sequencing, ChIP-seq

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Use immunocytochemistry to test for pluripotency markers on the protein level: Validate the presence of pluripotency cell surface proteins TRA-1-60, TRA-1-81, SSEA-3, and SSEA-4

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Single cell analysis within tissues

Analyse cell heterogeneity within a tissue, compare healthy and diseased tissues

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Labeled thymidine diluted in frequently dividing progenitors, but retained in infrequently dividing stem cell candidates