Compare density gradient centrifugation, magnet-activated cell sorting
(MACS), and fluorescence-activated cell sorting (FACS) in the isolation of pure stem cell populations from a heterogeneous suspension
Density gradient centrifugation is currently considered the gold-standard method for achieving the highest-purity exosome samples, as they remove non-specifically bound proteins from vesicles
Compare density gradient centrifugation, Magnet-activated cell sorting (MACS), and Fluorescence-activated cell sorting (FACS) in the isolation of pure stem cell populations from a heterogeneous suspension. Discuss the advantages and disadvantages of each technology as well as the emerging (recent) methods in stem cells separation.
Isolation of organelles is accomplished by cell membrane lysis and density gradient centrifugation to separate organelles from contaminating cellular structures. Intact nuclei and organelles have distinctive sizes in mammalian cells, enabling them to be separated by this method.
Density gradient centrifugation is currently considered the gold-standard method for achieving the highest-purity exosome samples, as they remove non-specifically bound proteins from vesicles
Compare density gradient centrifugation, Magnet-activated cell sorting (MACS), and Fluorescence-activated cell sorting (FACS) in the isolation of pure stem cell populations from a heterogeneous suspension. Discuss the advantages and disadvantages of each technology as well as the emerging (recent) methods in stem cells separation.
Isolation of organelles is accomplished by cell membrane lysis and density gradient centrifugation to separate organelles from contaminating cellular structures. Intact nuclei and organelles have distinctive sizes in mammalian cells, enabling them to be separated by this method.
Analytical Ultracentrifugation of protein.DiNa Amin
Ultracentrifuge is a high-speed centrifuge for separating microscopic and sub-microscopic materials to determine the sizes and molecular weights of colloidal and other small particles.
Introduction
Definition
Components of centrifuge
Types of centrifugation
Application
Conclusion
References
A centrifugation is a technique for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.
What are microbiological techniques ? what techniques or methods are used to detect microorganism. phase contrast mircroscopy , gel electrophoresis , flow cytometry , cell counter method and other.
Analytical Ultracentrifugation of protein.DiNa Amin
Ultracentrifuge is a high-speed centrifuge for separating microscopic and sub-microscopic materials to determine the sizes and molecular weights of colloidal and other small particles.
Introduction
Definition
Components of centrifuge
Types of centrifugation
Application
Conclusion
References
A centrifugation is a technique for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.
What are microbiological techniques ? what techniques or methods are used to detect microorganism. phase contrast mircroscopy , gel electrophoresis , flow cytometry , cell counter method and other.
Stem cells and nanotechnology in regenerative medicine and tissue engineeringDr. Sitansu Sekhar Nanda
Alexis Carrel, winner of the Nobel Prize in Physiology or Medicine in 1912 and the father of whole-organ transplant, was the first to develop a successful technique for end to end arteriovenous anastomosis in transplantation.
Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
The presentation gives a basic understanding of the principle of FACS, instrumentation, interpretation of results, applications, how to do cell-cycle analysis using FACS and various troubleshooting tips.
Dear students I hope this PPT finds you well. This presentation is all about the laboratory instrument centrifuge and its working called centrifugation. A centrifuge is a laboratory device used to separate fluids, gases, or liquids of different densities by spinning them at high speeds. It operates on the principle of centrifugal force, causing heavier particles or substances to move outward and settle at the bottom while lighter ones move toward the top. This separation process is employed in various fields such as medicine, biology, chemistry, and industry for tasks like separating blood components, purifying samples, and separating mixtures based on density variances. Centrifuges come in various types, including microcentrifuges, ultracentrifuges, and refrigerated centrifuges, each designed for specific applications requiring different speeds and capacities.Types of centrifuges and their functions include:
Microcentrifuge:
Function: Used for small-volume samples (typically ranging from 0.2 mL to 2.0 mL) in molecular biology, biochemistry, and clinical applications. It separates cellular components, proteins, DNA, and RNA.
Clinical Centrifuge:
Function: Primarily used in medical laboratories for processing blood, urine, and other bodily fluids. It separates components like red blood cells, plasma, and serum for diagnostic purposes.
Refrigerated Centrifuge:
Function: Similar to standard centrifuges but equipped with cooling systems to maintain low temperatures during separation. Ideal for samples sensitive to heat, like enzymes or biological materials.
Ultracentrifuge:
Function: Operates at extremely high speeds, separating particles at molecular levels. Used for studying macromolecules, lipoproteins, and subcellular particles.
High-Speed Centrifuge:
Function: Used in research labs for general separation tasks, capable of higher speeds than standard centrifuges. It's versatile and employed across various scientific disciplines.
Differential Centrifuge:
Function: Separates particles based on their sedimentation rates and size differences. It's used to isolate specific components from complex mixtures.
Preparative Centrifuge:
Function: Focuses on large-scale sample separation for purification purposes. It's employed in industrial settings for isolating biomolecules or other substances at higher capacities.
Centrifuges find applications in various fields such as molecular biology, biochemistry, medicine, pharmaceuticals, and industrial processes. Their versatility in separating substances based on density variances makes them invaluable tools in scientific research, diagnostics, and manufacturing processes.
ADAPTIVE SEGMENTATION OF CELLS AND PARTICLES IN FLUORESCENT MICROSCOPE IMAGEJournal For Research
Understanding the mechanisms of cell motility and their regulation is an important challenge in biomedical research. The ability of cells to exert forces on their environment and alter their shape as they move is essential to various biological processes such as the immune response, embryonic development, or tumor genesis .Recent technological advances in con-focal fluorescence microscopy have given researchers the opportunity to investigate these complex processes in vivo. However, they also lead to a tremendous increase in the amount of image data acquired during the studies. Therefore, the analysis of time-lapse experiments relies increasingly on automated image processing techniques. Namely, there is a high demand for fast and robust methods to help biologists to quantitatively analyze time-lapse image data. The potential of the proposed tracking scheme and the advantages and disadvantages of both frameworks are demonstrated on 2-D and 3-D time-lapse series of rat adipose-derived mesenchymal stem cells and human lung squamous cell carcinoma cells, respectively. The crucial tasks are, in particular, segmenting, tracking, and evaluating movement tracks and morphological changes of cells, sub-cellular components and other particles.
EVs have been previously classified into three main subtypes
based on their cellular origin, size, or biogenesis.
This includes (1) exosomes (30–150 nm) with an endocytic
origin, (2) microvesicles (100–1000 nm) formed by
budding of the plasma membrane, and (3) apoptotic bodies
(500 nm–2 μm) derived from dying cells
Based on new guidelines and the fact that determining
the exact biogenesis pathway of EV is still considered difficult,
use of a more general term of EV is recommended.
Moreover, for identifying EV subtypes, use of more operational
terms which refer to either their physical characteristics
such as size, density, biochemical composition,
descriptions of conditions or cell of origin is suggested.
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
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A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
for Anti-inflammatory, Antiulcer, Anticancer, Wound healing, Antidiabetic, Hepatoprotective, Cardio protective, Diuretics and
Antifertility, Toxicity studies as per OECD guidelines
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
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Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
1. TOPIC
Compare density gradient centrifugation, magnet-activated cell sorting
(MACS), and fluorescence-activated cell sorting (FACS) in the isolation of
pure stem cell populations from a heterogeneous suspension.
Figure 1: Cell separation using density
gradient centrifugation.
Figure 2: Cell separation using
magnetic-activated cell sorting (MACS).
Figure 3: Cell separation using
fluorescence-activated cell sorting
(FACS).
2. Introduction
Why do we need to isolate stem cell from heterogenous suspension?
- To get pure stem cells.
How do we isolate them ?
Physical Parameters
Affinity
Size
Density
Chemical
Magnetic
Electrical
3. Density Gradient Centrifugation
Separation of components of a sample on the basis of their density, in a density gradient, in a centrifuge,
according to the centrifugal force they experience.
2 principle types
Rate zonal centrifugation Isopycnic Separation
● Separation of cells/particles based on the
differences in size, shape & density.
● involves carefully layering a sample
solution on top of preformed liquid density
gradient
● centrifuged until the desired degree of
separation is achieved
● Time dependent
● Separation of cells/particles solely by their density, not
by shapes, sizes & time
● Particle size only affect the rate at which particles
move until their density is the same as the
surrounding gradient medium.
● Used to separate particles of similar size but of
different density.
4. Rate zonal centrifugation Isopycnic Separation
Sample is layered as a narrow zone on the top of a
density gradient
Under centrifugal force, particles move at different
rates depending on their mass
Starting with a uniform mixture of sample and
density gradient.
Under centrifugal force, particles move until their
density is the same as the surrounding medium
Density Gradient Medium
Selection
Ideal density gradient media properties:
1. Sufficient solubility to produce the
range of densities required.
2. Does not form solutions of high
viscosity in the desired density
range.
3. Is not hyperosmotic or
hypoosmotic when the particles to
be separated are osmotically
sensitive.
4. Does not affect the biological
activity of the sample.
5. Non-toxic and not metabolized by
cells.
6. Does not interfere with assay
procedure or react with the
centrifuge tube.
7. Easily removed from the purified
product.
8. Autoclavable.
9. Low cost.
Eg: CsCl (isopycnic separation of DNA),
sucrose (rate-zonal separation of DNA),
Ficoll (separation of cells and subcellular
fractions
Gradient medium: to provide a
gradient of viscosity which improves
particle resolution while stabilizing the
column from convection currents
Gradient medium: density of gradient
media is higher than that of the
particles
6. Magnet Activated Cell Sorting
Utilization of microbeads that detect specific antigens and bind to them,
separation is then carried out by subjecting the sample to a magnetic field.
MicroBeads
● Superparamagnetic particles of approximately 50 nanometers in diameter
● Detect specific antigens and bind to them
● Conjugated to monoclonal antibodies
● Biodegradable
MACS Separators
● Powerful permanent magnets that induce a high-gradient magnetic field within
MACS Columns
MACS Columns
● MACS separation process occurs within the MACS Columns.
7. How Does It Work ?
Magnetic Labelling
Indirect labelling
Magnetic Separation Elution of labelled cell
fraction
Negative selection
● Magnetic field ON
● Untagged cells will elute
out.
● Cell of interest retain on
column.
Positive selection
● Magnetic field OFF
● Tagged cells will be eluted out
Direct Labelling
9. Fluorescence Activated Cell Sorting
Fluorescence-activated cell sorting is a specialized
type of flow cytometry.
Fluorescence-activated cell sorting able to sort
cells of heterogeneous suspension into different
containers according to
-light scattering
-fluorescent characteristic of each cell.
10. How Does It Works ?
1. Treat cells with fluorescent antibody
marker.
2. Cell mixture flow in stream and leave
nozzle as droplet
3. Laser beam strikes
4. Charge given to cells
5. Pass through electrically charged plate
6. Cells are separated
13. Emerging Method
Methods Aqueous Two Phase System SELEX Microfluidics
Principle - polyethylene glycol (PEG) (upper
phase) and dextran (lower phase) for
centrifugation
- resulting target cells form sediment
bands at the interface of the two
phases
- uses RNA, ssDNA, or
modified nucleic acids as
aptamers to selectively
capture target cells with
their high affinity
- Aims at miniaturization
- Mimic in vivo
microenvironment for cell
differentiation
- Chip based
Method - Uses temperature sensitive polymer
poly(N-isopropylacrylamide)
(PNIPAAm) which soluble at 20°C but
precipitate at 32-35°C
- Conjugated antibodies recognize
specific stem cells, capture and
precipitate out through switching of
temperature
(Beili.Z & Shashi.K, 2013)
- Incubate stem cells with
aptamers and remove
unbound aptamers
- The bound aptamers are
subsequently released from
surfaces of the stem cells
and are then further
amplified by RT-PCR for
SELEX cycle
- Antibodies have been
immobilized onto the luminal
surface of a parallel array of
hollow fibers
- Detachment of target cells
was performed in fluid flow
with a pre-defined shear
stress.
(Menachery. A et al, 2017)
14.
15. Density Gradient Centrifugation
Why this method ?
- Most simplified and cost effective.
- Label free from magnetic particles or antibodies.
- Ready to use (STEMCELL Technologies Inc., 2012).
Conclusion
16. Why this method ?
- Widely used in clinical settings for large scale processing. Due to its faster separation
compared to FACS.
- More cost effective compared to FACS (Pierzchalski et al. 2013).
Magnetic Activated Cell Sorting (MACS)
17. Fluorescence Activated Cell Sorting (FACS)
Why this method ?
- High sensitivity and precision compared to MACS (Zhu, B and Murthy, SK, 2013).
- Better separation of populations using antibodies (Flow Cytometer Facility, n.d.)
18. Conclusion
Which method to use ?
Most efficient Most simplified
Ready to use
High specificity
Generally, affinity-based approaches are most efficient & reliable, due to high
specificity. (Zhu, B and Murthy, SK, 2013)
Cost effective Precision
19. References
Asami, M, Higuchi, S, Shibata, N & Agata, K, 2006, ‘Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting’, Vol. 48, no. 6, pp.
371-380.
Catherine, M., Brian T, F. and Timothy C, F. 2010, Fluorescence-Activated Cell Sorting for CGMP Processing of Therapeutic Cells. 1st ed. [ebook] Sparks: BD
biosciences, p.7, viewed 20 May 2017,
<https://www.researchgate.net/profile/Timothy_Fong2/publication/228470167_FluorescenceActivated_Cell_Sorting_for_CGMP_Processing_of
_Therapeutic_Cells/links/55f6ef6e08ae07629dbb159e.pdf>
Flow Cytometer Facility, n.d., ‘FAQs for cell sorting’, viewed on 24 May 2017,
<https://med.virginia.edu/flow-cytometry-facility/resources/faqs/faqs-for-cell-sorting/>
Handgretinger, R, Lang, P, Schumm, M, Taylor, G, Neu, S, Koscielnak, E, Niethammer, D, L & Klingebiel, T, 1998,’Isolation and transplantation of autologous
peripheral CD341 progenitor cells highly purified by magnetic-activated cell sorting’, vol. 21, pp. 987-993.
Miltenyibiotec.com. (n.d.). MACS manual Cell Separation Columns - Miltenyi Biotec, online, viewed on 22 May 2017,
http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/manual-cell-separation/columns.aspx
Miltenyi Biote, (n.d.) MACS Technolody Golden Standard in cell separation, viewed on 24 May 2017,
http://www.dartmouth.edu/~dartlab/uploads/MACS_Technology_Flyer.pdf
Oscar, Tom, Chen, WM, Lee, KD, Hsieh, SL & Chen, TH, 2003, ‘Isolation of multipotent mesenchymal stem cells from umbilical cord blood’, viewed on 20 May
2017, <http://www.bloodjournal.org/content/bloodjournal/103/5/1669.full.pdf?sso-checked=true>
20. Pierzchalski, A, Mittag, A, Bocsi, J, & Tarnok, A, 2013, ‘An Innovative Cascade System for Simultaneous Separation of Multiple Cell Types’, viewed on 24 May 2017,
<http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074745>
Shi, S and Gronthos, S, 2003, ‘Perivascular Niche of Postnatal Mesenchymal Stem Cells in Human Bone Marrow and Dental Pulp’, vol. 18, no. 4, pp. 696-704.
STEMCELL Technologies Inc., 2012, ‘How to Use SepMate™ to Isolate PBMCs from Whole Blood in Just 15 Minutes’, viewed on 20 May 2017,
<https://www.stemcell.com/how-to-use-sepmate-to-isolate-pbmcs-from-whole-blood-in-just-15-minutes.html>
Uchida, N, Buck, DW, He, D, Reitsma, MJ, Masek, M, Phan, TV, Tsukamoto, AS, Gage, FH, & Weissman, IL, 2000, ‘Direct isolation of human central nervous system
stem cells’, vol. 97, no. 26, viewed on 20 May 2017, <http://www.pnas.org/content/97/26/14720.abstract>
Zhu, B and Murthy, SK, 2013, ‘Stem Cell Separation Technologies’, vol. 2, no. 1, pp. 3-7.
References