Question

Looking for a little insight, Thanks in advance

Create a 5 minute video using screencast-o-matic describing:

What flow cytometry is

What the principles of flow cytometry are when it switches to Fluorescence Activated Cell Sorter (FACS)

What data one might interpret from scatter plots generated

What the applications are in immunology

Answer 1

Flow Cytometry

Flow cytometry is the quantitative analysis of cells (“flow” refers to cell movement, “cyto” means cells,” metrics” refers to measurement). Flow cytometry is a laser-based technique, that utilizes light to enumerate cell count and profile, from a heterogenous sample mixture.

Flow cytometry is an analytic cell-biological or biophysical technique, that is used for the analysis of expression and identification of cell surface or intra or inter-cellular molecules, determines viability of cells, cell type, cell sorting, assessing purity or percentage of a specific type of cells, biomarker detection and characterization of other multi-parameters of a type or different types of cells. It has gained importance in clinical research, study, medical analysis, immunological analysis, cancer study (especially blood cancer).

The basis steps of flow cytometry include:

1. Cell samples are made to flow through a narrow channel or suspended through stream of fluids. Through this light (laser) is passed.

2. The cells then refract or emit light, different wavelengths (absorbed spectra and emitted spectra), which are detected using sensors (spectroscopy, mass spectrometry).

3. The data acquired though these sensors are interpreted and analyzed accordingly (usually through computer programming).

FACS:

Fluorescence-activated cell sorting or sorter (FACS) is a specialized flow cytometry technique (benchmark of Becton, Dickinson, and company), which allow the separation or sorting of cells, from a heterogenous mixture, based on fluorescence properties and light scattering of cells.

The technique involves:

1. Cells are labelled with fluorescent dyes.

2. Cell suspension are allowed in a narrow nozzle, with vibrating mechanism to allow single droplets, and cell separation according to diameter.

3. The cells passes a fluorescence measuring detector.

4. A charge-based separation is also devised and a electrostatic detection is inserted.

Interpretation of data:

When a cell passes through the laser beam, a signal is generated. This signal generated, from each point is obtained as pulse in the detector. Certain pulses, which are below the threshold levels are ignored and not considered.

Quantification if obtained by plotting these pulses as a function of time.

In scatter plots, two axes of graph are represented as two detectors and the cells are represented as dots. It represents 2D or two parameters. Cells will scatter light when they pas through the laser beam. The level or degree of cell scattering is proportional to cell size. This is depicted as forward scattering. The other components of cells, such as organelles, granules, compartments, etc., generate side scattering.

The scatter plot analyzes, the number of experiments performed, average data, spread of data.

Application in Immunology:

1. Counting number of B-cells or T-cells in a sample of blood.

2. Detecting subpopulation of WBC and their activation in immune response.

3. Analysis of antibody labeled cells.

4. Analysis of CD surface proteins by directing antibodies.

5. Identifying different CD clusters by fluorescence dye tagging.