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- 1. Addis Ababa University College of Health Sciences Department of Medical Biochemistry Cell sorting & Cell fractionation techniques By Ousman Mohammed (ID no. GSR/7263/15) 1
- 2. Outline • Introduction to cell sorting techniques • Functions of cell sorting • Preparation of samples for cells sorting • Cell sorting techniques • Cell fractionation • Summary • References 2
- 3. Objectives After completing this session, learners should be able to: • Describe some common cell sorting techniques • Explain the functions of cell sorting • Differentiate the different types of cell sorting techniques • Understand advantage and disadvantages of different cell sorting techniques • Explain about cell fractionation steps 3
- 4. Introduction • Cell sorting is the process through which a particular cell type is separated from others • It is based on its differences in – Size, morphological parameters, viability protein expression • Applications including research, diagnosis, and therapy 4
- 6. Cont… • Capture of circulating tumor cells (CTCs) from blood • Isolation of immune cells from peripheral blood • Isolation of WBC from tissue • Conduct molecular analysis of a single cell type, including RNA expression and epigenetic analysis 6
- 7. Preparation of samples for cells sorting A successful sort results in good purity and yield of the target population and is dependent on these things: Cell harvesting and preparation Cell staining and fluorochrome choice Proper controls Sample and sorting conditions Sample collection conditions 7
- 8. Preparation cont… Isolating cells from tissues or adherent cultured • Disrupting the extracellular matrix holding the cells together using mechanical force and/or proteolytic enzymes • Incomplete dissociation of cell clumps can lead to inefficient labeling of the target cells • Processing method should not affect cell surface epitopes, as this may negatively impact both cell isolation and downstream functional analyses 8
- 9. Strategies for cell sorting Positive selection • The cell type of interest is targeted by the removal mechanism and retained for downstream analysis • Targeting a cell surface marker with a mAb or ligand and directly labeling desired cells for selection • Isolated cells are bound by Ab or particles 9
- 10. Strategies cont… Negative selection • Performed by removing several cell types to leave one cell type untouched • Involve labeling unwanted cell types for removal with Ab targeting specific cell surface proteins • Antibody cocktail target all unwanted cells but not desired cells • Protocols are faster and easier • E.g. Removing all cells except for T cells or the removal of all cells except for B cells from samples 10
- 11. Cont… 11
- 12. Techniques of cell sorting • Major cell sorting techniques includes: • Fluorescence activated cell sorting (FACS) • Magnetic-activated cell sorting (MACS) • Buoyancy-activated cell sorting (BACS) 12
- 13. Fluorescence activated cell sorting (FACS) • One of the most commonly used methods in cell sorting • This method relies on cell suspensions which contain a decent number of fluorescent target cells • FACS utilizes flowcytometry to separate cells based on morphological parameters & protein expression • Interestingly, these systems are able to isolate even rare and single cells 13
- 14. FACS cont… • Three main components: – Fluidics – Optics – Electronics • This involves encapsulating cells into small liquid droplets which are selectively given electric charges and sorted by an external electric field 14
- 15. How Does FACS Work? • Antibodies match with antigens on the surface of targeted cell • Targeted cell is labeled with fluorescent marker and mixed into the cell sample • One at a time, cells flow through a point of analysis where they are hit by a laser beam • Cells are then emptied into containers based on their fluorescence 15
- 16. FACS cont… • Cells are injected into a “flow cell” which is located in the optical path of a light source • Cells are made to move (or focused) in single file using liquid pressure through a small (50-300 µm) orifice = hydrodynamic focusing 16
- 17. Fluorescence activated cell sorting (FACS) 17
- 18. FACS cont.. 18
- 19. Advantages and Disadvantages FACS • Sort single cells • Sort complex cell types with multiple markers at higher purity • The ability to separate cells based on their – surface markers as well as size and granularity allow for more in-depth isolations • However, Bulky volume, expensive price • Complicated operation • Annoying sample preparation cell staining (fluorescence-labeling) 19
- 20. Magnetic Activated Cell Sorting (MACS) Based on the attachment of small, inert, supra- magnetic particles to mAbs specific for antigens on the target cell population The cells are separated by magnetic particles through an antibody interaction with surface markers of the targeted cells Cells labelled to these antibody-bead conjugates are then separated via a column containing a ferromagnetic matrix 20
- 21. How Does MACS work? • Magnetic beads coated with Ab or enzymes associated with surface markers of the targeted cells • Magnetic beads label the cells with recognizable surface markers • The targeted cells attach to the magnetic beads and are magnetized to the column walls while non- targeted cells flow through the sample column • Cells isolate between targeted and non-targeted 21
- 22. Advantages and Disadvantages • MACS is 4-6 times faster than FACS – This allows researchers to run a higher quantity of samples in a shorter time • On the other hand, MACS can also be extremely harsh on fragile cells • The magnetic nature of the process can cause damage to the target cell membrane 22
- 23. Buoyancy-activated cell sorting (BACS) • Is a cell selection process that involves sorting cells with buoyant microbubbles • Microbubbles labeled with antibodies specific to the target cells • When mixed into the sample, the microbubbles bind to the target cells • Due to the augmented buoyancy force, the microbubbles float to the surface, separating the target cells 23
- 24. BACS cont… • The microbubbles are first mixed with the cell sample, and bind to the target cells • After binding with the target cells, the microbubbles float to the top. The target cells float with them, leaving behind the non-target cells • In the final step, the microbubbles and target cells are removed from the top of the sample 24
- 25. BACS cont… 25
- 26. BACS cont… A quick alternative to more traditional cell sorting technology It has proven ability to reduce cell sorting time and is easy to use Takes an average of 10-30 minutes for cell selection Relatively inexpensive 26
- 27. Single-cell sorting • Based on intracellular and extracellular properties – Every cell is individually analyzed • Enable understanding of cellular properties that may be obscured or non-evident • Results in highly enriched cell populations that are more homogeneous than those obtained via bulk sorting methods • Microfluidic device with micro-channel valves to trap a single cell in a chamber • E.g. FACS 27
- 28. Bulk cell sorting • Highly rely on cell characteristics like size and density • Results enriched cell populations that are less homogeneous than those obtained via single cell sorting methods • All of the target cells are collected in one sweep • E.g. Filtration, centrifugation, and magnetic cell sorting 28
- 29. Cell fractionation • Is the process used to separate cellular components while preserving individual functions of each component • This is a method that was originally used to demonstrate the cellular location of various biochemical processes • Other uses of subcellular fractionation is to provide an enriched source of a protein for further purification, and facilitate the diagnosis of various disease states • Cell fractionation involves 3 steps: Extraction, Homogenization and Centrifugation 29
- 30. Cont… Extraction • The first step toward isolating sub-cellular structure • Suspension should be keep in an appropriate medium • Cells or tissues suspend in a solution with an adequate pH and salt content, typically an isotonic solution with a pH of 7.0 30
- 31. Cell fractionation cont… Homogenization • Tissue is typically homogenized in a buffer solution • Mechanisms for homogenization include: – Grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thawing • The samples are then kept cold to prevent enzymatic damage • It involves grinding of cells in a suitable medium in the presence of certain enzymes with correct pH, ionic composition, and temperature 31
- 32. Cell fractionation cont… Purification • Is achieved by differential centrifugation results in the sequential separation of organelles according to their density 32
- 33. Summary • Cell sorting is a process to isolate one or more specific cell populations from a heterogeneous mixture of cells • They are separated majorly based on differences in cell size, morphology & surface protein expression • They have important applications in research and as therapeutics • Common methods of cell sorting techniques include FACS, MACS • Cell fractionation is the process used to separate cellular components 33
- 34. References • Steven A Soper, Malgorzata A Witek (2020). "Cell Separations and Sorting". Anal Chem. 92 (1): 105–131 • Zhu B and Murthy SK (2013). Stem cell separation technologies. Curr Opin Chem Eng 2, 3–7. • Dirican, Enver Kerem (2012), "Magnetic-Activated Cell Sorting of Human Spermatozoa", Practical Manual of in Vitro Fertilization, Springer New York, pp. 265–272 • Chen YW et al. (2017). A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat Cell Biol 19, 542–549. • Ma, Z et al.( 2017). "Fluorescence activated cell sorting via a focused traveling surface acoustic beam". Lab on a Chip. 17 (18): 3176–3185. 34
- 35. THANK YOU! 35
Editor's Notes
- Cell sorting is the process through which a particular cell type is separated from others contained in a sample on the basis of its physical or biological properties, such as size, morphological parameters, viability and both extracellular and intracellular protein expression. The homogeneous cell population obtained after sorting can be used for a variety of applications including research, diagnosis, and therapy For many cell sorting methods, fluorescently labeled antibodies, which only bind to specific cell types or cells in certain stages of cellular development, are being applied to identify the cells of interest and thus to distinguish target cells from unwanted cells.
- In oncology and cancer research, it has been shown that tumors not only contain tumor cells, but also immune cells are invading the tumor tissue. Moreover, the tumor cells also vary amongst each other regarding their genomic information due to acquired cancer mutations and thus contribute to tumor heterogeneity. Molecular pathologists are therefore interested in cell sorting technologies for liquid biopsies to be able to make a better informed diagnosis.
- Capture of circulating tumor cells (CTCs) from blood Isolation of immune cells (T Cells, B Cells, etc.) from peripheral blood Isolation of WBC from tissue Preparing a sample of blood separated from plasma Separation of pathogenic bacteria from food Conduct molecular analysis of a single cell type, including RNA expression and epigenetic analysis
- The goal of preparing tissues for cell sorting is to maximize the yield of functionally viable, dissociated cells. Unfortunately, these types of samples often contain a high percentage of dead cells and debris as a byproduct of the dissociation process, which interferes with the quality of the sort and the resolution of the target population. Preparing Adherent Cultured Cells: Adherent cultured cells are most commonly removed from the culture substrate by treatment with trypsin. Trypsin formulations and conditions vary depending on the cell type but incubating cells with a trypsin concentration too high for too long will damage cell membranes and kill the cells. In addition, trypsin can alter cell surface antigens and therefore alter binding of detection antibodies used to identify target populations. To inactivate trypsin use of a trypsin inhibitor such as Soybean trypsin inhibitor is better than serum, as serum adds back divalent cations that facilitate cell adhesion/aggregation. To inhibit cell aggregation, EDTA can be added as a divalent 2 cation chelator as described in Section 4 (Sample conditions for sorting). Cell Enrichment/Depletion and Red Blood Cell (RBC Lysis) : Whether to perform enrichment or depletion depends on the frequency of the target population and the specimen type. Depletion of unwanted cells that constitute a larger population in your sample and RBC lysis cut down the sorting time and increase the efficiency of rare event sorting. Depletion/Enrichment can be done by using either magnetic bead based technology (such as AutoMACS) or by using density gradient centrifugation. Reagents like Lympholyte (Cedarlane) can be used for enriching lymphocytes from non-lymphoid organs by density gradient. Fluorochrome Selection: Consider doing a preliminary analysis of your experiment before bringing your cells for sorting. Generally, “positive” populations that are dim and only minimally separated from a slightly dimmer "negative" population will lead to a poor analysis and/or a poor sort. A thoughtful balance of fluorochrome brightness with cellular marker abundance is important for optimal resolution of cell populations. Please refer to the brightness chart on our website and the BioLegend website for more details on multicolor panel design. Spectral Overlap: Another important consideration in panel design is the amount of spectral overlap between fluorochromes. Spectral spill-over from a very brightly stained channel into a detector that requires high-sensitivity can be a real problem. This problem of overlapping emission spectra can be minimized with the use of one of the many “Spectra Viewers” available online. Please see the links for fluorochrome panel design software and tools in the “Related Resources” tab on our website. Blocking Non-specific Binding: An ideal antibody would have a high affinity to only one, specific cellular epitope. Unfortunately, non-specific binding can be a problem even when using a correctly-titrated antibody. In these cases, a blocking reagent is needed. Usually, a blocking reagent contains a high concentration of species-specific immunoglobulin that can bind to the Fc-receptors that are often responsible for the non-specific binding of the staining antibody. Specific antibodies to Fc receptors can also be purchased and have been used successfully, see the eBioscience website or the Innovex website for more information. Titrating Antibodies: The optimal concentration for antibody labeling is when the ratio of antibody to antigen reaches a point of saturation. Too low and there will not be enough antibody to saturate all of the high-affinity binding sites; this will limit brightness, make small pipetting errors significant and make quantitative conclusions about cytometric data suspect. Too high an antibody concentration will waste reagent, and could actually lower the signal-to-noise ratio due to high non-specific binding to low-affinity sites. 3.CONTROLS: Unstained and Single Stained Control(s): Unstained controls are essential for determining background fluorescence and single stained controls are used to calculate the correct compensation values before a sort. Incorrect compensation can result in the wrong cells being sorted. Please bring an unstained/ nonfluorescent control AND single stained controls for each fluorochrome/dye/fluorescent protein being used in your experiment to each and every sort appointment. Fluorescence Minus One Controls or FMO’s: A fluorescence minus one (FMO) control contains all fluorochromes of the multicolor cocktail except one. They represent the combinatorial background fluorescence from other channels into the channel of interest and aid in setting sort gates properly. Many researchers are resistant to using FMO controls on a routine basis because they consume cells, reagents and time, but in many cases they are needed to validate the gating strategy of a reagent panel particularly if you are using the panel for the first time. 4.SAMPLE AND SORTING CONDITIONS: A successful sort is dependent on appropriate sample and sorting conditions! The presorted cells should be in a buffer that maintains live, clump-free cells in suspension for the duration of the sort. In addition, it is vital to select the correct instrument settings for the cell type being sorted. Selecting Sorting Conditions: The type of sorter, sheath pressure and nozzle size can greatly impact cellular function and experimental outcome for downstream applications. Selecting the correct parameters is largely dependent on cell size and morphology in the sorted sample. Basic Cell Sorting Buffer: •1x PBS (Ca/Mg++ free) or HBSS (preferred) •0.5-2% BSA or up to 2% heat-inactivated FBS [dialyzed against Ca/Mg free PBS] •25mM HEPES pH 7.0 5.SAMPLE COLLECTION CONDITIONS: Optimal Collection Media: The collection media is the post-sort solution that receives the droplets containing target cells in sheath from the sorter. If the sheath is PBS, the calcium chloride in most culture media is not compatible with the phosphate component of the PBS leading to precipitation of calcium phosphate crystals when a large number of cells are sorted. The optimal collection media will depend on the downstream experiments planned for the sorted cells, but below are some suggestions. Fetal Bovine Serum 100% to 50% in PBS OR Your own culture media with antibiotics OR PBS if collecting cells for RNA or DNA OR Lysis buffer from RNA isolation kit (e.g. buffer RLT from Qiagen kit) Lysis buffer dilution could be a problem depending on number of cells collected and nozzle size.
- Ca2+/Mg2+ - free buffers: helps to reduce cell aggregation Use BSA (0.1 - 1%) or dialyzed FBS (1 - 5%) Use a minimal amount of BSA to decrease autofluorescence and to increase population resolution. Avoid non-dialyzed FBS, as it facilitates cell-cell adhesion by replacing Ca and Mg Add EDTA (2 - 5 mM) → helps prevent cell adhesion Add 10 – 25 mM of HEPES to improve pH stability. Add DNAse I (25 – 50 ug/mL) and 5 mM of MgCl2 → digests free DNA released by dead cells There are several enzymes commonly used in tissue dissociation protocols: Collagenase can hydrolyze collagen and is widely used for isolating cells from animal tissues. Hyaluronidase is often used in combination with collagenase and catalyzes hydrolysis of 1,4-β-Dglycosidic linkages. DNase is added to cell suspensions to minimize cell clumping due to DNA released by damaged cells. Elastase is used to digest tissues containing high amounts of elastin. Trypsin is a serine protease with a specificity for peptide bonds and is often combined with other enzymes (e.g. elastase and/or collagenase) for tissue dissociation.
- 1. Positive selection The cell type of interest is targeted by the removal mechanism and retained for downstream analysis Performed by targeting a cell surface marker (CD4, CD8, etc.) with a monoclonal antibody or ligand and directly labeling desired cells for selection. Antibody cocktail targets a unique surface marker on the target cells. Isolated cells are highly purified. Isolated cells are usually bound by antibodies.
- 2. Negative selection Similar to depletion, the negative cell separation approach is when several cell types are removed to leave one cell type untouched. Involve labeling unwanted cell types for removal with antibodies or ligands targeting specific cell surface proteins Antibody cocktail target all unwanted cells and do not target desired cells. Protocols are faster and easier with minimal sample manipulation Isolated cells are not bound by Antibodies or magnetic particles example is the depletion of all cells except for T cells or the removal of all cells except for B cells from samples like whole blood or bone marrow. . Depletion A single cell type is targeted and removed from a biological sample For example, the removal of red blood cells from peripheral blood mononuclear cells (PBMCs) is completed via depletion
- One of the most commonly used methods in cell sorting is FACS (fluorescence activated cell sorting). This method relies on cell suspensions which contain a decent number of fluorescent target cells. Thus, target cells need to be specifically labeled with a fluorescent dye via antibodies or they need to express fluorescent proteins to be detected by the FACS cell sorter. A similar method to FACS is Magnetic Activated Cell Sorting (MACS), where antibody-coated magnetic beads specifically bind to the target cells to be able to separate them with a strong magnet from non-labeled cells. In addition to FACS and MACS cell sorters, there are several other cell sorting methods based on microfluidics or on filter technologies. These methods are ideal for cellular enrichment of one certain cell type or of a specific cell population. They also require a relatively high number of cells and suspension volume. In contrast, cell picking and micromanipulation systems, which are typically installed on microscopes, are highly selective and can work with small amounts of sample. Intriguingly, these systems are able to isolate even rare and single cells. Initially, these devices for microscope cell sorting heavily relied on the manual handling capabilities of the individual users. Today, fully automated cell picking instruments are available that allow for selective cell picking and medium-throughput cell sorting.
- Target cells need to be specifically labeled with a fluorescent dye via antibodies or they need to express fluorescent proteins to be detected by the FACS cell sorter One of the most commonly used methods in cell sorting is FACS This method relies on cell suspensions which contain a decent number of fluorescent target cells
- Fluorescence activated cell sorting has several systems that work together to achieve successful sorting of events of interest. These include fluidic, optical, and electrostatic systems.
- Cells are separated by their fluorescent properties or by scattered light – either by cell size or a scatter that indicates granularity. Check out our article on FACS vs. flow cytometry to get a better understanding of how they differ. Fluorescence-activated cell sorting, also known as FACS, is a type of flow cytometry using fluorescent labeling to target and isolate groups of cells. After being run through a cytometer, they are sorted based on their physical characteristics (size, granularity, etc). Common practices of FACS include hematopoiesis, oncology, and stem cell research. How Does FACS Work? FACS is cell separation based on the cell surface markers and sorts the biological mixture in two or more containers based on light scattering. Proteins and carbohydrates give each cell unique surface phenotypes. The cells are given specific antibodies which are associated with the cell surface antigens. Cells are then targeted by those antigens.
- The fluidics system is the lifeblood of the flow cytometer. It is responsible for aligning the cells in single file in the core stream, and passing them through the interrogation point for data collection. Without consistent sample introduction and correct cell alignment, the data spread will be large and your confidence in the data will be reduced. Flow cytometers use hydrodynamic focusing or acoustic-assisted hydrodynamic focusing to control the flow of the cells through the interrogation point, allowing more precise data collection.
- Fluorescence-activated cell sorting (FACS) analyzes each cell individually. Extremely Powerful Technique that can provide a large amount of information at once. Ideal Quantification method for multiplex immunoassays. Uses Flow Cytometry and Fluorescent Probes to sort heterogeneous mixtures of cells. The cells are incubated with fluorophore-labeled antibodies before the sort. The antibodies are specific to surface antigens on target cells. Each antibody has a different emission wavelength and is uniquely identifiable. One method of precisely labeling antibodies with fluorophores is on-bead labeling with Protein A. After incubation with the labeled antibodies, the cell solution is sent through the flow cytometer. This machine guides the solution through a micron-sized nozzle one cell at a time. Each cell moves through a laser excitation area, where the laser excites the fluorophores bound to the cell surface. The fluorescent emission is recorded, and the cell is directed either into a collection container or a discard container according to user-defined parameters
- These last considerations are of particular importance when cell sorting is used for clinical applications, for example cell therapy and should therefore be performed under Good Manufacturing Practice (GMP) conditions. Moreover, flow cytometry cell sorters are complex instruments that are generally used only by well-trained staff in flow cytometry facilities or well-equipped laboratories and, since they are normally big in size, it is not always possible to place them inside a biological safety cabinet. Therefore, it is not always possible to ensure sample sterility and, since the fluidic systems can be cleaned but it is not single-use, there is the possibility of cross-contamination among samples. Another aspect to be considered is that droplet generation inside the instrument could lead to aerosol formation that are hazardous for the operator when using infectious samples. FACS is much more versatile than MACS. The ability to separate cells based on their surface markers as well as size and granularity allow for more in-depth isolations. This trait has made FACS a standard in many research labs, but that doesn’t mean there are no limitations. Flow cytometers, which are machines required to perform FACS, are very costly. Additionally, proper training for operating the equipment can be time consuming. Although sample sizes can be large, the amount of time it takes to run a single sample falls around 2-3 hours. The high quantity also increases the likelihood of contamination in the sample. When such a high volume of cells is flowing through the laser, it’s easy for cells to get sorted incorrectly or misidentified. FACS is much more versatile than MACS. The ability to separate cells based on their surface markers as well as size and granularity allow for more in-depth isolations. This trait has made FACS a standard in many research labs, but that doesn’t mean there are no limitations. Flow cytometers, which are machines required to perform FACS, are very costly. Additionally, proper training for operating the equipment can be time consuming. Although sample sizes can be large, the amount of time it takes to run a single sample falls around 2-3 hours. The high quantity also increases the likelihood of contamination in the sample. When such a high volume of cells is flowing through the laser, it’s easy for cells to get sorted incorrectly or misidentified. FACS is much more versatile than MACS. The ability to separate cells based on their surface markers as well as size and granularity allow for more in-depth isolations. This trait has made FACS a standard in many research labs, but that doesn’t mean there are no limitations. Flow cytometers, which are machines required to perform FACS, are very costly. Additionally, proper training for operating the equipment can be time consuming. Although sample sizes can be large, the amount of time it takes to run a single sample falls around 2-3 hours. The high quantity also increases the likelihood of contamination in the sample. When such a high volume of cells is flowing through the laser, it’s easy for cells to get sorted incorrectly or misidentified. FACS is much more versatile than MACS. The ability to separate cells based on their surface markers as well as size and granularity allow for more in-depth isolations. This trait has made FACS a standard in many research labs, but that doesn’t mean there are no limitations. Flow cytometers, which are machines required to perform FACS, are very costly. Additionally, proper training for operating the equipment can be time consuming. Although sample sizes can be large, the amount of time it takes to run a single sample falls around 2-3 hours. The high quantity also increases the likelihood of contamination in the sample. When such a high volume of cells is flowing through the laser, it’s easy for cells to get sorted incorrectly or misidentified. FACS is much more versatile than MACS. The ability to separate cells based on their surface markers as well as size and granularity allow for more in-depth isolations. This trait has made FACS a standard in many research labs, but that doesn’t mean there are no limitations. Flow cytometers, which are machines required to perform FACS, are very costly. Additionally, proper training for operating the equipment can be time consuming. Although sample sizes can be large, the amount of time it takes to run a single sample falls around 2-3 hours. The high quantity also increases the likelihood of contamination in the sample. When such a high volume of cells is flowing through the laser, it’s easy for cells to get sorted incorrectly or misidentified.
- MACS (Magnetic Activated Cell Sorting) Superparamagnetic nanoparticles are made of a core of iron oxide, typically magnetite (Fe3O4), which is not innately magnetic, but becomes magnetized by an applied magnetic field. These particles or beads are coated with silica or a polymer surface to prevent clumping, and a well-chosen coating also provides a rich surface for the covalent attachment of functional groups and antibodies. Provide high degree of specificity to a cell enrichment protocol. The attachment of antibodies provides the superparamagnetic particles with specificity. The functionalized particles are incubated with the target cell solution, and the cells with surface antigens complementary to the antibodies will bind to form a cell-bead conjugate. The conjugates are enriched by magnetic cell separation. is a good choice when specificity is desired. is rapid and efficient. By applying a magnetic field to the matrix, the beads stick to the matrix inside the column and the bead-carrying cells are held back from passing through.
- Magnetic beads coated with Ab or enzymes associated with surface markers of the targeted cells are added to the sample Magnetic beads label the cells with recognizable surface markers The targeted cells attach to the magnetic beads and are magnetized to the column walls while non-targeted cells flow through the sample column Cells isolate between targeted and non-targeted This can take on a positive or negative cell selection approach This solely depends on whether the targeted cells stay attached to the walls of the column or pass through
- Specific, Rapid, and Efficient when care is taken to develop and finely tune a sorting strategy. When working with cells of a higher rarity, MACS can make it difficult to safely extract a high percentage of the desired substance
- Utilizes glass microbubbles labeled with antibodies specific to the target cells. When mixed into the sample, the microbubbles bind to the target cells. Due to the augmented buoyancy force, the microbubbles float to the surface, separating the target cells. Buoyancy Activated Cell Sorting (BACS) is a cell selection process that involves sorting cells with buoyant microbubbles. These miniature gas-filled bubbles allow for a gentle separation of extremely small cells or particles from a larger mixed population. This is a simple and inexpensive cell sorting method compared to the others that require various equipment such as magnetic columns. The BACS method has the ability to reduce cell sorting time and costs, which is considered a major benefit for many users. Another advantage this process has over others is the ability to decrease exposure of cell samples to lasers and magnets that could potentially diminish the quality of the overall final product.
- Microbubbles are mixed with cell samples, allowing them to identify and bind to targeted cells and separate from non-targeted cells Once targeted cells have been secured, microbubbles float to the top Targeted cells are carried through leaving behind the non-targeted cells Targeted cells are collected from top of the sample to be isolated or removed from the enriched sample left behind
- Single-cell sorting provides a method for sorting a heterogeneous mixture of cells based on intracellular and extracellular properties Single-cell methods enable understanding of cellular properties that may be obscured or non-evident. There are several methods for sorting single cells: The microraft array provides a rapid, cost-effective method for isolating cells, analyzing cells over time, and generating clonal populations with the unique ability to monitor all intra- and extracellular properties.[45] This system is ideal for both adherent and non-adherent cell types. A single-cell method to observe the response to an external stimulus (in this case, cellular response to a ligand) was studied using a microfluidic device with micro-channel valves to trap a single cell in a chamber. A 23-valve system was used for actuation, and fluorescent dye was used in stimulus-response imaging.[46]
- Highly rely on cell characteristics like size and density Results enriched cell populations that are less homogeneous than those obtained via single cell sorting methods All of the target cells are collected in one sweep. Example: Filtration, Centrifugation, and Magnetic Cell Sorting
- Fractionation of the cells into subcellular compartments enables protein enrichment and is essential to accurately determine the localization of specific proteins, which is the first step towards understanding the function of a protein in the cell. Cell fractionation allows you to study the many components of a cell separately. For example, you can see which organelles produce the most energy after the organelles are isolated. Cell fractionation is a procedure for rupturing cells, separation and suspension of cell constituents in isotonic medium in order to study their structure, chemical composition and function. Is the process used to separate cellular components while preserving individual functions of each component This is a method that was originally used to demonstrate the cellular location of various biochemical processes Other uses of subcellular fractionation is to provide an enriched source of a protein for further purification, and facilitate the diagnosis of various disease states
- Extraction The first step toward isolating sub-cellular structure To ensure that organelles and biomolecules retain their biological function under mild conditions (known as cell-free systems) before being used, you have to maintain isotopic constraints At 0-40°C, the cells or tissues suspend in a solution with an adequate pH and salt content, typically an isotonic solution of sucrose (0.25 mol/L) with a pH of 7.0 Suspension should be keep in an appropriate medium
- Homogenization Tissue is typically homogenized in a buffer solution that is isotonic to stop osmotic damage Mechanisms for homogenization include: grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thawing, and ultra-sound The samples are then kept cold to prevent enzymatic damage It is the formation of homogenous mass of cells (cell homogenate or cell suspension) It involves grinding of cells in a suitable medium in the presence of certain enzymes with correct pH, ionic composition, and temperature For example, pectinase which digests middle lamella among plant cells.
- What equipment is used for cell fractionation? The central piece of equipment in cell fractionation is a centrifuge. A centrifuge is a piece of equipment that spins rapidly and thus adds a centripetal force on the object that is spinning. Filtration This step may not be necessary depending on the source of the cells. Animal tissue however is likely to yield connective tissue which must be removed Commonly, filtration is achieved either by pouring through gauze or with a suction filter and the relevant grade ceramic filter Purification Purification is achieved by differential centrifugation – the sequential increase in gravitational force results in the sequential separation of organelles according to their density