Cell based methods including flow cytometry Flashcards

Question

name some applications of The Flow Cytometer

Answer 1

Phenotype of cells, surface molecules, intracellular cytokine staining, cell signaling molecules, cell sorting, cellular pH, calcium flux assays, apoptosis analysis, cell cycle analysis, cell proliferation, cellular transport assays, drug uptake/efflux assays, transfection efficiencies, phagocytosis assays, etc.

Answer 2

Fluidics =To focus the cells for interrogation by a laser Optics = To generate and collect the light signals (scatter and fluorescence) Electronics = To convert the optical signals to electronic signals and digitize them for computer analysis (PMTs)

Answer 3

-Its relative size (Forward Scatter—FSC) -Its relative granularity or internal complexity (Side Scatter - SSC)

Answer 4

A dot plot in the context of flow cytometry is a graphical representation that shows the relationship between two parameters (e.g., forward scatter and side scatter, or two different fluorescence channels) for each cell analyzed. Each dot on the plot represents a single cell, allowing researchers to visualize and interpret complex data from heterogeneous cell populations. x- axis is forward scatter (represents size) y-axis is side scatter(represents granularity) Double-positive cells (expressing both markers) will have high values on both axes and will appear in the top-right quadrant. Double-negative cells (expressing neither marker) will have low values on both axes and will appear in the bottom-left quadrant

Answer 5

components of molecules which absorb light and emit fluorescence They typically contain aromatic groups

Answer 6

Absorption of a photon raises the electron from ground state to an excited state. Emission of photons as the electron returns from an excited state to ground state gives rise to the fluorescence.

Answer 7

used for marking cell surface and intracellular antigens (Common labels: FITC, PE, APC)

Answer 8

two covalently attached fluorescent molecules (donor and acceptor) e.g. Pe/Cy7 * behave as a unique fluorophore - excitation properties of donor, emission properties of acceptor

Answer 9

* PMT (photomultiplier tubes) signals are converted to digital information and processed in a computer. * The information can be viewed as histograms, dotplots, density plots or contour plots.

Answer 10

Negative Population in the bottom left square Single Positive PE Population in the top left square Double Positive Population in the top right square Single Positive FITC Population in the bottom right square

Answer 11

Fluorescence from one channel, i.e. FITC

Answer 12

Annexin-V staining is a commonly used method to detect early apoptosis in cells. Annexin-V is a protein that binds specifically to phosphatidylserine (PS), a phospholipid that is normally found on the inner leaflet of the plasma membrane. During apoptosis, PS is translocated from the inner to the outer leaflet of the plasma membrane, exposing it to the external environment. Annexin-V can bind to PS with high affinity, making it a useful marker for detecting apoptotic cells

Answer 13

vital dyes that bind to nucleic acids, such as DNA, in cells. They are commonly used in flow cytometry and fluorescence microscopy to distinguish between viable, apoptotic, and necrotic cells based on membrane integrity. These dyes can only penetrate the plasma membrane when the membrane integrity is compromised, such as in the later stages of apoptosis or during necrosis. In viable cells with intact membranes, these dyes are unable to enter the cells. * Bind to nucleic acids * Can only penetrate the plasma membrane when membrane integrity is breached, as occurs in the later stages of apoptosis or in necrosis.

Answer 14

Live Cells: Cells that are negative for dead cell markers and exhibit normal metabolic activity are considered viable. Dead Cells: Cells that stain positive for dead cell markers or show decreased metabolic activity are considered non-viable

Answer 15

Cells that are negative for both Annexin V and 7AAD/PI have no indications of apoptosis. PS translocation has not occurred and the plasma membrane is still intact.

Answer 16

Cells that are Annexin V-positive and 7AAD/PI – negative. PS translocation has occurred, but the plasma membrane is still intact

Answer 17

Cells that are positive for both Annexin V and the 7AAD/PI are either in the late stages of apoptosis or are already dead. PS translocation has occurred and the plasma membrane integrity is lost.

Answer 18

it helps to correct for spectral overlap and ensure accurate interpretation of fluorescence signals in flow cytometry

Answer 19

Multiplex analysis of cells in suspension. The number of lasers in modern instruments are increasing. Advanced conventional instruments: 5 lasers -> 21-28 parameters to be measured in parallel. Fluorochromes continously being developed which enables increased complexity. You can analyse with gates and subgates, giving detailed information on a sample. Excitation – what lasers you have will affect your choice. Emission spectras have to be considered when multiplexing. Should overlap as little as possible

Answer 20

The different fluorochromes have unique spectra. However, the leak of the red spectra into the filter that is supposed to measure the yellow spectra is large -> problem in conventional flow cytometry (Compensation will not be enough!) Spectral provides spectral unmixing! Higher multiplexing possible with spectral!

Answer 21

These factors are indeed critical considerations in flow cytometry experiments: Compensation: Correcting for spectral overlap ensures accurate interpretation of fluorescence signals when using multiple fluorophores. Unspecific Staining: Differentiating between specific and nonspecific binding is essential for obtaining reliable results. Titration of antibody concentrations and appropriate blocking can help minimize nonspecific staining. Isotype Controls: Isotype controls are crucial for distinguishing specific antibody binding from background signal. They involve using antibodies of the same isotype as the primary antibody but with irrelevant specificity. Autofluorescence: Cell fluoroscence even when no fluorescent tag or stain is added. Autofluorence comes from normal cell components, such as riboflavin and flavoproteins

Answer 22

* Allows separation of a cells of interest from a hetero-geneous population. * Charges are applied to cells of interest. * Deflection plates attract cells with opposite polarity

Answer 23

– allow the cytokine profile of individual cells to be determined 1. Activated T cells are treated with an inhibitor which blocks protein export allowing cytokines to accumulate in the ER 2. the cell in fixed and permeabilized with mild detergents. 3. Cytokine- specific antibodies penetrate the cell to bind the intracellular cytikines.

Answer 24

- Synthetic nucleoside that is an analog of thymidine. - Incorporated into the newly synthesized DNA of replicating cells - Can be measured by antibodies towards BrdU (intracellular staining) - > Frequency of proliferating cells (As an alternative to measuring Thymidine incorporation – but here we need a flow cytometer)

Answer 25

Beads with emission wavelength of 650 nm. Different amounts –> different levels of signal -> bead (and thus analyte) identification Labeled antibodies that specifically capture a particular molecule The captured analyte is detected by PEconjugated antibodies (max ca 575 nm)' + Small sample sizes + Very sensitive + Short assay time + Easy to use + Multiplex + Does not require special instrument (if FACS available) -Expensive

Answer 26

1.Add sample to specific antibody-bead (serum/plasma or cell supernatant) 2.wash 3. Add biotinylated detector antibody 4. wash 5. Add Streptavidin-PE 6. wash 7. Read in Luminex machine Total time 3.5 hrs + Small sample sizes + Very sensitive + Short assay time + Easy to use + Multiplex , >100 analytes - Expensive - Requires special instrument

Answer 27

Detect and quantify T-cells that are specific for a given antigen – cytomegalovirus peptide MHC class I (HLA) molecule They bind directly to T cell receptors of a particular specificity, determined by the MHC allele and peptide combination. MHC class I tetramers – -for studies of CD8+ T cell responses MHC class II tetramers – - for studies of CD4+ T cell responses