Category: Flow Cytometry Technique Master Certification Course

This Master certification course on Flow Cytometry will develop practical skills and theoretical knowledge of the modern instrumentation, tools and techniques used to examine cells by flow cytometry. You are encouraged to make links between the various sessions and your research to contextualise knowledge and to assist in unifying the theoretical and practical components and applying them to your research in future .

In this course you will learn about –

• workings of a flow cytometer
• set-up and calibration
• compensation
• fluorescence and fluorochromes
• pre-analytical methods
• quality control
• immunodetection
• data analysis using different softwares
• strategies for the analysis of peripheral blood samples
• applications in immunology.

In addition, you will learn about Multicolor panel design, harmonisation and standardisation of different flow cytometers, advanced data analysis using different softwares, hematopoiesis and analysis of stem cells, immature, and mature cell populations, specific applications in hematology, detailed case studies.

Areas of study will include application and theory of analytical and sorting flow cytometry and the uses of these instruments and techniques in the research laboratory environment. The particular focus of the practical tasks will be tailored to your research field. This may include but need not be limited to immunology, microbiology, pharmacy, plant or zoology studies.

Benefits 

After completing this Master certification course of Flow cytometry , you are able to / has knowledge as well as skills related to different aspects .

Knowledge

You gain knowledge in:

• Understanding for basic applications for Flow Cytometry
• which and how many different fluorochromes one can use on a given flow cytometer
• evaluate the quality of flow cytometry data

Skills

You will develop specific skills after completing this particular course:

• Plan and perform basic experiments using Flow Cytometry
• Quality control generated flow cytometry data
• Present flow cytometry data in a scientific way

Competence 

• Understanding of basic flow cytometry
• Understanding as to why to use flow cytometry in a certain research question
• Interpret basic flow cytometry data

How Important is the Knowledge Of Flow Cytometry in Research Career?

Flow cytometers were initially used in clinical laboratories to obtain complete cell count. Their reliability and ease of handling increased its demand in research. Now, flow cytometry is an inevitable technique used in biological research for its wide range of applications, especially in the fields of cell biology, immunology, molecular biology, and cancer biology.

Flow cytometric data can be spotted in almost every scientific publication related to cell biology and this demonstrates its worldwide utilization and importance. Moreover, a large proportion of scientific publications related to immunology and biomedical sciences report flow cytometry data. Nowadays, most of the leading universities and biomedical research institutes possess their own flow cytometers, despite its high cost. They are also used for diagnosis and research in hospitals and medical centers worldwide.

The major diagnostic applications of flow cytometer include cell cycle, ploidy, and surface analysis of cancer. They are also used in the study of cell surface markers for leukemias and lymphomas, which could prove useful in their diagnosis. Flow cytometry is the method of choice for measuring CD4 lymphocyte levels in the blood to monitor the progression of AIDS and response to treatment. Less expensive methods are yet not available. In research, clinical applications, and clinical trials, cell sorting turn out to be highly important.

If you are someone aspiring for a research career in biosciences, flow cytometry is one such technology you must be skilled in. Be it an interview for a research position or an actual research project you are handling, you are expected to have full information on flow cytometry, including the working principle and data analysis. To conclude, flow cytometry is indispensable in the field of biology.

Role in Career Advancement 

Flow cytometry Master certification course is very essential to all those students or individuals who are planning to have a career in this field in future. So many features are there related to this course that will help you to earn extra points for your resume. The popularity of this analytical tool can be estimated by its range of uses. It has been used for cell counting, cell sorting, measuring cell size and volume, characterizing different cell types in a mixed cell population, analyzing both cell surface and intracellular molecular expression and assessing the purity of isolated subpopulations of cells. Principally checks the fluorescence intensity generated by fluorescent-labeled probes associated with proteins, lipids, DNA or any other specific cellular molecules.

The diversified application of flow cytometry has made it the new-age analytical tool. The popularity of flow cytometry could be well estimated by the growth in the global antibody market and reagent market, which in 2017 amounted to about $945 million and $2.8 billion, respectively. With an increase in demand for primary antibodies, the major application has been observed in the field of flow cytometry.

First of all many job openings are available in research in labs, academic sector and commercial sector as research fellow- JRF/SRF, research associate, research scientist, Post-Doctoral Fellow (PDF) and lab technician in universities, research institutes, and hospitals . Many pharmaceutical and clinical companies also employ persons with expertise in flow cytometry specifically those associated with antibody maker development, designing disease targets and drug targets such as cancer, involved in the diagnosis, clinical trials and quality control and assurance. Moreover, other job opportunities are also available apart from direct research or machine operation.

Some of the companies frequently hiring experts in flow cytometry are

• Boehringer Ingelheim India Pvt Ltd
• Thermo Fisher Scientific
• Syngene International Ltd
• AstraZeneca Pharma India Ltd
• Iqvia
• Pfizer Inc
• Nestle Ltd.
• Apex Services
• BD India Pvt Ltd
• MilliporeSigma, Merck Ltd
• Phenomenex Inc.
• Cancer Genetics Inc.
• Averinbiotech Pvt Ltd
• Becton Dickinson India Pvt Ltd.
• Thyrocare Technologies Ltd and many more.

Flow cytometry is a complex field that draws people from diverse scientific backgrounds. Whether you’re an immunologist or aquatic ecologist, researcher or clinician, we hope this guidebook will help you in your journey of discovering the powerful technology and applications of flow cytometry.

Flow cytometry is the science of measuring physical and chemical properties of live cells or other biological particles as they pass in a fluid, single-cell stream through a measuring apparatus. In the most common scenario, one or more lasers interrogate each particle and, at a minimum, the system measures the degree and direction of scattered light — indicators of the particle’s size, shape and structure. If particles have been stained with one or more fluorescent dyes — known as fluorochromes — the light source excites these dyes to provide additional biological information about each particle, such as metabolic activity, DNA content and the presence of specific surface and intracellular markers. Precise optical and electronic elements collect the fluorescent pulses and scattered light, convert them into digital values and send them to a computer for analysis. Some flow cytometers are also equipped to identify and sort user-specified particles into collection vessels. High-performance cell sorters can routinely reach rates of 70,000 cells per second.

The unique power of flow cytometers is that they can rapidly and quantitatively measure multiple simultaneous parameters on individual live cells and then isolate cells of interest. Additionally, the sensitivity and throughput rates achievable by high-performance commercial instruments enable detection of extremely rare populations and events (frequencies below 10-6), such as stem cells, dendritic cells, antigen-specific T cells and genetic transfectants.1 As a result, applications for flow cytometers continue to grow.

In addition to traditional immunology and pathology applications involving particles such as lymphocytes, macrophages, monocytes and tumor cells, flow cytometers are widely used in conjunction with fluorescence-based protein reporters, such as green fluorescent protein (GFP). In this arena, flow cytometers can monitor both transfection efficiency and protein expression levels. They also can detect fluorescence resonance energy transfer (FRET), which provides information about molecular interactions, protein structure and DNA sequence .

Ongoing development efforts in the flow cytometry industry are aimed at automation and laboratory integration. Input/output robotics, pushbutton operation and automated sample preparation will increase throughput rates and make the technology more accessible to a wider user base, as new fluorescent dyes and creative screening approaches expand applications into the proteomic arena. Eventually, software advances will seamlessly network instruments into comprehensive analytical and diagnostic systems, and the industry may marry its technology with imaging and microfluidics. Obviously, this is only a very superficial discussion of the field of flow cytometry. It is intended merely to set the stage for the rest of the publication, which will introduce you to more details about the technology and applications of flow cytometry.

How Does Flow Cytometry Work?

The basic principle of flow cytometry involves the passage of cells in a stream through a laser beam. The cellular particles could be fluorescently labeled and then excited by the laser to emit light at varying wavelengths. Each cell passing through the cytometer scatter some of the laser light and emit fluorescence following the excitation. The instrument then measures the fluorescence as well as the scattered light to determine the amount and type of cells present in the population.

Several parameters are simultaneously measured by a flow cytometer;

1. Forward scatter intensity (FSC): FSC is directly proportional to the cell diameter.
2. Side scatter intensity (SSC): SSC represents the granularity of the cell.
3. Fluorescence intensity: Light emitted at various wavelengths is measured.

FSC alone is quite useful in excluding cell debris, cell aggregates, and dead cells. It is sufficient in distinguishing lymphocytes from granulocytes or monocytes in blood samples. The granularity of living cells like dendritic cells can be determined from SSC. Fluorophores coupled to antibodies are used to mark specific subpopulation of cells, and thus to quantify, sort, or study those cells from a heterogeneous cell population.

The ability of flow cytometers to measure the flourescense has many more applications in biology.

Applications of Flow Cytometry:

The research applications of flow cytometry include:

– Immunophenotyping: Flow cytometry is most commonly used in immunophenotyping, which detects and quantifies a specific population of cells in a heterogeneous sample, usually lymph, blood, or bone marrow. Hematological malignancies like leukemia and lymphomas can be diagnosed in clinical labs by immunophenotyping the sample.

– Transfection efficiency: By using fluorescent protein (i.e. GFP) as a marker, flow cytometry can be used to determine the transfection efficiency. The flow cytometer can determine the percentage of successfully transfected cells within a transfected cell population.

– Apoptosis: Apoptosis, the programmed cell death is a normal process among the eukaryotic cells. Cells can die due to a variety of reasons and apoptosis in cells can be detected by several flow cytometric methods. Cells are usually stained with 7ADD or Annexin V to determine apoptosis.

– Cell cycle analysis: By fixing the cells and then staining the cellular DNA with dyes like ethidium bromide, propidium iodide, and DAPI, flow cytometry can be used to quantify the cells in G1, S, G2 and M phases. Since the dye binds to DNA, the fluorescent signal is directly proportional to the amount of DNA present inside the cell, which varies with different phases of the cell cycle.

– Cell proliferation: Cell proliferation is often studied in cell biology to measure cellular metabolic activity in response to stimuli like cytokines, media components, and growth factors. By labeling the cells with carboxyfluorescein succinimidyl ester (CFSE), a cell membrane fluorescent dye, the flow cytometer can measure the proliferation of cells. As the cell divides, the dye is passed on to the daughter cells, each getting half the original dye. Researchers can calculate the proliferation by measuring the reduction in fluorescence signal.

– Cell sorting: A particular subset of cells can be sorted from a heterogeneous population based on desired parameters.

– Membrane potential: DiOC2 is a fluorescent membrane-potential indicator dye which exhibits green fluorescence in all bacterial cells. In cells with larger membrane potentials, the dye becomes more concentrated and shifts to red. The green or red bacterial populations can be easily distinguished using a flow cytometer.

– Live/dead bacteria discrimination: Using the combination of two dyes thiazole orange and propidium iodide, the flow cytometer provides a reliable method to discriminate live and dead bacteria. The efficacy of antibiotics in killing microbes can be determined this way.

While this was just a glimpse, Dive Into The Course To Learn More In Detail.