Can A Cell Sorter Be Used To Filter Blood Cells

Cell Sorter

In summary, the prison cell sorter takes labeled cells, pressurizing them up to 70+ PSI, pass them through the laser interrogation point, vibrate them at rates of 90kHz, as they reenter atmospheric force per unit area, where they receive an electric charge, and laissez passer through an electric field only to striking the surface of a drove medium and remarkably virtually of these cells survive.

From: Pathobiology of Human Affliction , 2014

Microfluidic devices for stem cell analysis

D.-Chiliad. Kang , ... West. Zhao , in Microfluidic Devices for Biomedical Applications, 2013

Magnetic-activated stalk cell sorting

MACS, one of the nearly popular conventional cell isolation methods, has recently been developed in microfluidics to isolate rare cells. Tan and colleagues get-go introduced micro-magnetic separators for stalk jail cell sorting (Fig. 11.24) (Tan et al., 2005). A 3D mixer was integrated in a microfluidic channel to reach lamination with 180-degree rotations and rapid mixing between cells and magnetic beads. To isolate the target cell from the mixture, magnetic beads conjugated with CD31 antibodies were used to remove CD31 + endothelial cells with an external magnetic field. Up to 90.2% of hMSCs were isolated and recovered. In addition, Souse and colleagues introduced a two-inlet/two-outlet microfluidics device to isolate mouse mESCs using super-paramagnetic particles. To isolate specific embryonic antigen 1 positive (SSEA-ane +) mESCs from a heterogeneous population of mESCs, anti-SSEA-i antibodies were conjugated onto super-paramagnetic beads and mixed with the jail cell mixture. Once the mixture was injected into the microfluidics channel and the magnetic field was applied, SSEA-i + mESCs were deviated from the direction of laminar menstruum according to their magnetic susceptibility and were thus separated from SSEA-1⁻ mESCs.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780857096975500116

Menses CYTOMETRY

Charlotte Nexmann Jacobsen , Mogens Jakobsen , in Encyclopedia of Food Microbiology, 1999

Jail cell Sorting

In a flow cytometer equipped with a cell sorter it is possible to dissever and collect cells of interest ( Fig. i). Flow sorting is a combination of one or more of the flow cytometric parameters measured on cells and concrete separation of exactly those cells having a specific characteristic. The more parameters used to describe the cells to exist sorted, the more specific and effective is the resultant sorting. The principle of flow sorting is based on the deflected droplet method. Originally the cells were sorted according to their book, but now the cells can exist sorted on behalf of whatsoever of the parameters, offered past menses cytometry. The cells pass through the flow sleeping accommodation where they are analysed and leave the sleeping room equally aerosol. A voltage is applied to the droplets containing the leaner to be sorted, leaving them with a positive or negative accuse. The droplets are passed through an electrostatic field, where they are deflected according to their charge and collected. Uncharged droplets pass through the electrostatic field to the waste matter container. Cell sorting has been used to split up Staphylococcus aureus from E. coli with final cell purities of 95% or greater. Starved cells of Micrococcus luteus were sorted into groups of feasible, fallow and dead cells co-ordinate to their ability to bind the dye rhodamine 123, whereas the β-galactosidase activity was used for sorting viable bacteria and yeasts with very high efficiency.

Cell sorting seems to exist an effective method of separating cells of interest occurring at low levels in heterogeneous microbial populations.

Read full affiliate

URL:

https://www.sciencedirect.com/science/article/pii/B0122270703006656

Flow Cytometry Software

J.P. McCoyJr., in Pathobiology of Homo Disease, 2014

Background

The advent of menstruum cytometry harkens dorsum nearly half a century with prototype jail cell sorters and analyzers being adult by several groups. Past the early on 1970s, the starting time commercial cytometers, such as the ICP 11 (Partec and Phywe) and the cytofluorgraph (Ortho Diagnostics), appeared on the market place, and the utilise of flow cytometry for medical and life science research started to become accepted by wide range of scientists. However, if one recalls the status of computers, peculiarly, personal computers, in those days, information technology speedily becomes apparent that the computational aspects of flow cytometers were lagging. (The Apple I reckoner was non introduced until 1976, the Apple Two in 1977, and the IBM 5150 PC until 1981.) Indeed, it was not unusual in the early on days of period cytometry for Polaroid cameras to exist used to take pictures of oscilloscope screens to capture data. The rapid improvement in computing applied science in the 1990s and early xx-first century more than kept pace with developments in flow cytometry, and as a result, data capture and analysis were not a serious constraint in most areas of menses cytometry. But recently, new cytometry technologies have been introduced, such equally imaging flow cytometry, high-dimensional catamenia cytometry, and mass spectroscopy flow cytometry, which learn such big amounts of data in a single file that an boilerplate desktop calculator would not suffice for both capture and assay.

The early days of menstruum cytometry, as in the early on days of the personal computer, saw many formats for capturing, and thus analyzing, data. In some instance, data were simply stored as histogram displays of the actual data, only more than commonly, data were collected and stored as 'listing fashion' files. List style files store the raw data on a prison cell-by-cell ground for all cellular parameters collected in the society in which the information are acquired. Thus, a list is made for each parameter for every cell. To bring order out of this anarchy, in 1984, Tater and Chused proposed a file standard (FCS 1.0) termed 'flow cytometry standard' (*.fcs). Their proposal stated that such files would have a minimum of three segments: TEXT, Information, and Analysis. The Data File Standards Commission of the Society for Analytical Cytology (now the International Society for Analytical Cytometry) afterwards modified this structure in several means and included a quaternary segment, the HEADER. The revision (FCS two.0) proposed by the commission contained a detailed data file structure standard for flow cytometry and became widely adopted by manufacturers of commercial cytometers. 2 subsequent revisions to this format have been published, one in 1997 (FCS three.0) and most recently in 2010 (FCS 3.one).

The wide acceptance of file standards for period cytometry data not only permitted cross platform sharing of data but likewise fostered the development of 'tertiary-party' software programs for the analysis of data as well as some file management or manipulation. This was a tremendous asset to the menses cytometry customs equally this non only brought down the cost of such programs but as well fostered extended data analysis of period cytometry files since this could now be performed 'off-line' from the acquisition cytometer.

In this article, we will review types of information display, software programs for diverse applications, file management and utility software, and new approaches for analyzing and understanding high-dimensional immunophenotyping. Finally, mention volition be made of ongoing efforts to automate data analysis. This is not meant to be an encyclopedic review of all programs, by and nowadays, for the analysis of flow cytometric information, just rather volition provide an overview of concepts and approaches to analysis.

Read full affiliate

URL:

https://www.sciencedirect.com/scientific discipline/article/pii/B9780123864567071033

Applied Aspects of Hematologic Stem Prison cell Harvesting and Mobilization

Scott D. Rowley , Michele 50. Donato , in Hematology (Seventh Edition), 2018

Collection of PBSCs by Apheresis

A number of apheresis devices are available for separating HSCs from the peripheral blood. The devices may be classified as continuous flow (due east.k., Fenwal CS3000, COBE Spectra, Spectra Optia, Fenwal Amicus) or discontinuous menstruum (e.thousand., Haemonetics family of equipment). Discontinuous-period devices have the advantage of requiring simply a unmarried venous access. Continuous-flow devices require two access lines for aspiration and return of claret, but they procedure much larger volumes of blood in a shorter flow of time. All apheresis devices collect HSCs. Continuous-flow devices are more than efficient in the collection of PBSCs and are, appropriately, preferred over discontinuous-menses devices (see box on Physician Prescription for Apheresis and Peripheral Blood Stem Prison cell Processing). The ideal apheresis device will take: a high efficiency of CD34⁺ cell collection with minimal contamination of the product past mature blood cells such as granulocytes, which complicate subsequent processing and tin can increase infusion toxicity; rapid blood processing with minimal anticoagulant apply reducing toxicity to the donor; and minimal depletion of platelets. The various apheresis devices, in general, accept similar efficacies in collecting CD34⁺ cells from the peripheral blood, simply may vary in processing time, and in last product volume and mature blood jail cell contagion. ¹⁰³

Doc Prescription for Apheresis and Peripheral Claret Stalk Cell Processing

The attending medico should be aware of apheresis and laboratory procedures in order to maximize the value of peripheral blood stem cell (PBSC) products. The choices of venous access, anticoagulant(southward), claret volume processed, target dose of CD34⁺ cells, and cryopreservation volumes tin can be individualized. Apheresis unit staff may inquire for guidance regarding pain medications, concurrent medications, and claret transfusions (although it is advisable to avoid transfusion during the apheresis procedure considering of citrate or other reactions, and because changes in the hematocrit may affect the efficiency of the collection). Adequate numbers of CD34⁺ cells can be nerveless for more than one cycle of chemotherapy. Information technology is important to communicate with the cryopreservation facility if PBSC components will be used to support more than one cycle of chemotherapy or if dimethyl sulfoxide toxicity is of business concern, in club to facilitate appropriate packaging of each product. Current cytometric techniques for quantification of CD34⁺ cells require at least 1 hour of processing, and then it may not be applied to prescribe the number of CD34⁺ cells to be frozen in each pocketbook. Nevertheless, information technology is possible to carve up the component into the number of bags equaling the number of predictable infusions so that equal numbers of CD34⁺ cells will be available for each.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780323357623000950

Hybridomas, T Cell

Shoichi Ozaki , in Encyclopedia of Immunology (Second Edition), 1998

Cloning

Cloning, the initiation of a cell line from a unmarried progenitor, tin exist achieved either in soft agar, past limiting dilution, or by using a cell sorter. Cloning past limiting dilution is performed by culturing hybridoma cells in 96-well plates at <1 cell per well together with feeder cells. Even on this condition, a significant proportion of wells receive two or more cells and hence cannot initiate a true clone; therefore cloning must be repeated to ensure the homogeneity of the hybrid lines. To verify the clonality it should exist demonstrated that their T jail cell receptors are homogeneous at the level of proteins, mRNA or genes.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B012226765600311X

Bacillus anthracis toxins

Shihui Liu , ... Stephen H. Leppla , in The Comprehensive Sourcebook of Bacterial Poly peptide Toxins (Quaternary Edition), 2015

Cell specificity and receptors

Nearly all types of eukaryotic cells possess receptors for PA. Specific bounden of radiolabeled PA to cells is easily demonstrated considering more 80% of total binding can be blocked past the add-on of nonradioactive PA. Binding can too be demonstrated in fluorescent cell sorters and by other means. Quantitative analysis of bounden data shows that cells possess a single grade of high-analogousness receptors, having clan constants of approximately one nM [164,165]. Unlike prison cell types have between 5000 and 50,000 receptors. Cells with about fifty,000 receptors include the L-6 rat myoblast and the man melanoma prison cell line LOX IMVI. The modest number of receptors, their sensitivity to trypsin, the low nonspecific binding, and the linear bounden curves suggested that the receptor was a single cell surface protein in the cells examined.

Chinese hamster ovary (CHO) cell mutants lacking functional PA receptors were obtained by the choice of mutagenized populations with PA and fusion proteins containing LFn and the catalytic domains of ADP-ribosylating toxins. No cross-resistance was found to other toxins, and all mutants mapped to the same complementation group [126,166]. This proved that the receptor gene in CHO cells was functionally hemizygous (i.e., a unmarried functional allele). CHO cells of this type provided the basis for an expression-cloning approach that successfully identified the receptor [thirty]. A retroviral expression library from HeLa cells was transfected into the receptor-deficient CHO cells, which were then sorted repeatedly for cells having enhanced receptor expression. The clone obtained independent complementary Deoxyribonucleic acid (cDNA) matching a previously described gene denoted tumor endothelial marker 8 (TEM8), which is highly expressed in endothelial cells of colorectal cancer tissues [167]. Subsequently, a closely related cistron designated capillary morphogenesis cistron 2 (CMG2) was besides shown to encode a functional receptor [31]. These genes are also annotated as ANTXR1 and ANTXR2, respectively. Through generating and analyzing TEM8- and CMG2-goose egg mice and TEM8/CMG2 double-nada mice, the authors' lab has demonstrated that CMG2 is the major physiologically relevant receptor in vivo, whereas TEM8 plays merely a minor part in anthrax infection and the toxins' activeness [168]. This is likely because CMG2 is widely expressed and has a 3–x fold higher affinity for PA than does TEM8. Although TEM8 was named based on its discovery as a protein highly expressed in tumor endothelium [167,169], the cDNA was later found to exist readily amplified by contrary transcriptase PCR from diverse tissues [168], arguing that TEM8 is also a wildly expressed gene. CHO cells and RAW264.7 cells express merely the CMG2 receptor, and HeLa cells express only TEM8, while well-nigh other cultured tumor prison cell lines express both receptors. Because TEM8/CMG2 double-null mice are completely resistant to challenge with extremely loftier doses of anthrax toxins [170], information technology is likely that only TEM8 and CMG2 function as anthrax toxin receptors in vivo.

CMG2 and TEM8 contain a domain similar the von Willebrand or I (inserted) domain that is characteristic of many integrins. The I domain contains a metallic ion dependent adhesion site (MIDAS) that is essential for binding to extracellular matrix molecules, such as collagens, that are the recognized ligands for these integrins. Mutagenesis of this site showed that it was required for PA bounden [thirty,171]. Mutagenesis of PA showed that the D683 side chain, previously proven to be important for PA part, is a potent candidate for a residuum that would interact with the metal ion bound at the MIDAS of the I domain. Purification of recombinant forms of the extracellular domains of TEM8 and CMG2 immune detailed report of their interaction with PA. A structure was obtained for the extracellular domain of CMG2 that was consequent with other I domain proteins [117]. Most important, structures were obtained for the complex of PA with CMG2 [117,137]. These structures demonstrated, somewhat surprisingly, that PA interacts with CMG2 not only through the surface of domain 4 to the MIDAS of CMG2 (with a surface interaction area of 1300 Åⁱⁱ every bit for other integrins), just also via the well-ordered loop (2β3−2β4) of PA domain ii to a CMG2 pocket (as the rest Glu-122 is located at the bottom of the pocket) side by side to the MIDAS motif. The large surface area of the interaction (about 1900 Å^two) explains the high affinity of PA binding to its receptors (Kd=0.2–i nM for CMG2, and Kd=3–ten nM for TEM8) [168,171,172]. The other important conclusion from the construction was that the interaction with receptors is predicted to prevent the movement of the PA domain 2 loop that occurs during the acid-induced insertion into cellular membranes. This brace or clamping action of the receptor imposes a requirement that the toxin reach an acidic endocytic compartment to undergo prepore-to-pore conversion [117,137].

PA spring to cellular receptors must be activated by proteolytic cleavage at aa 167 to larn the ability to oligomerize and to demark LF and EF (as discussed previously; also see Figure xiii.one). The cellular enzyme that most apace activates PA is furin [173]. However the cleavage site, RKKR, has more bones residues than the minimum furin recognition sequence RXXR, and appears to be susceptible to other cellular proteases. Thus, furin-deficient cells retain some sensitivity to wild-type PA but are totally resistant to PA mutants with an RAAR sequence. Information technology has been found that furin is the dominant activating enzyme for several other toxins and viruses, but that other cellular proteases can also perform the activation, although less efficiently. It is now articulate that PA can also be candy past unidentified proteases in the plasma of experimental animals, leading to circulating PA63 oligomers [174,175]. Thus, it is believed that PA proteolytic activation and prison cell-surface binding are independent processes, their rates dependent on the availability of cell-surface receptors and prison cell-surface and soluble proteases. The soluble PA63 oligomer and the effector proteins LF and EF may assemble a toxin complex before binding to cellular receptors [176,177].

It is also of interest that urokinase plasminogen activator and several matrix metalloproteinases, the proteases abundant on tumor cells, tin very efficiently activate PA mutants having appropriate target sequences substituted at aa 164 [132,178–180]. This implies that receptor-bound PA is either mobile in the lipid membrane or that any localization into membrane domains does not preclude contact with certain cell-surface proteases.

Read total affiliate

URL:

https://www.sciencedirect.com/science/commodity/pii/B9780128001882000136

Inside the Flow Cytometer: Hardware

T. Bushnell , J. Trotter , in Pathobiology of Human Illness, 2014

Fluidics

The fluidic components of the flow cytometer are designed to carry the labeled cells from the sample tube into the flow cell where the cells pass 1 or more interrogation points and complete their journeying either into a waste container or in the case of a cell sorter into a collection device. This process involves understanding two principles of fluid dynamics – laminar catamenia and hydrodynamic focusing.

Laminar flow, or streamline flow, describes when fluids flow in smooth parallel layers with no disruption between these layers. The particles move in sequence along the axis of the pipe, equally illustrated in the succeeding text. Additionally, considering of the increasing drag nearer to the sides of the pipe, the velocity of the fluid layers follows a parabolic arc, with the fastest catamenia in the centre of the innermost part of the cavalcade of liquid. In a menstruum cytometer, a carrier liquid, oft termed 'sheath,' surrounds the sample.

The cells are injected into the center of this sheath liquid with slightly more than force, inducing the sample to period into the period sleeping accommodation. This process of the cells beingness injected into the menstruum jail cell inside the sheath and spreading out single file in an organized style is termed hydrodynamic focusing. With the organization achieving laminar flow, the cells in the core spread out along the velocity axis, forming a 'single file' stream of entrained cells, which take been described equally beads on a cord appearance. This center stream where the cells and sample buffer are located is termed the 'cadre stream' ( Figure 2 ).

This can be visualized by imagining a wooden pencil, with the graphite in the center of the pencil. The wood of the pencil represents the sheath fluid, and the graphite core represents the cells spreading along the flow axis. It is important to notation that because of laminar flow, the sheath and the sample do non mix. In analytic cytometers, this means i could use h2o for the sheath fluid, although most laboratories apply some form of saline for sheath to proceed the refractive indices of sample and sheath equally close as possible, thereby minimizing any optical aberrations since refractive differences at the buffer interface tin act equally a lens. As long as the sheath and the sample are of similar densities and fluidic properties, no mixing occurs. Nevertheless, in the case of vastly different viscosities or buffer properties, such as when the sample contains a lot of protein, organic solvents (e.g., ethanol or methanol from fixation protocols) or may accept significant levels of detergents (from permeabilization protocols), this tin can negatively impact the fluid dynamics and issue in turbulent flow – meaning that cells will not pass the correct interrogation point or in an organized manner, resulting in poor data. The stream tin also be disrupted by concrete blockage. The most common causes of this are clogs caused by poorly prepared cells or air bubbles that have entered the menses jail cell. In the case of the former, proper cell preparation and the use of filters to removed clumped cells before loading the sample are the all-time solutions. Air bubbles, once lodged in the flow prison cell or fluid lines, often require more drastic measures, including running cleaning solutions (detergents and/or organic solvents) to disrupt the surface tension of the air chimera.

Cells are moved from point A to point B based on differential pressure between the sample and the sheath fluid. There are three major means to generate this differential pressure, that is, by a pressurized organization to push the sample ( Figure 3 ), a vacuum-operated organization to pull the sample, or using syringe-/pump-based systems to inject the sample ( Effigy 4 ). The almost mutual cytometer fluidic designs on the market are the pressurized based systems.

Every bit shown in the same figure, there are 2 different pressure regulators. The kickoff, which is kept at a constant pressure on the sheath fluid, sets the rate of flow for the organization. The 2d regulator controls the corporeality of sample by setting a differential pressure level on the sample. This force per unit area departure pushes the sample into the menses jail cell, where it is introduced into the sheath flow. The 2d regulator is what the end user adjusts to control the consequence rate of the system. Since the speed of the overall flow is fixed, past increasing the differential force per unit area, the merely mode for more events to pass the interrogation bespeak is for the size of the core stream to enlarge. Predictably, this has the end result of increasing the number of events passing the intercept point at a given fourth dimension. The downside of this procedure is increased variability of the data and more coincident events, both of which negatively impact the quality of the data, and this can be seen in Figure 5 . Best practices in running samples are to gear up the differential period rate equally low as needed to have a tight core stream close to the size of the particles being measured while achieving the desired number of events per second and to keep it at that flow rate; practice not change between samples only to increase event charge per unit. Adjusting sample concentration to increment event charge per unit significantly is preferred to simply increasing the differential sample catamenia.

It is worth noting that volumetric-based systems are typically smaller than differential pressure level system and are often quieter. Another reward of the volumetric system is that these systems accept intrinsic control over the absolute book of sample delivery, allowing for a precise prison cell count per unit book that is impossible in differential pressure level or vacuum organisation without the add-on of a command 'counting' dewdrop or volumetric sensor. Most cytometers are of the differential force per unit area blueprint, and some do provide optional volumetric sensors for the sample line, be they pressure level- or vacuum-based fluidics.

The eye piece of the fluidic components is the catamenia prison cell. This is where the sheath and the sample containing cells come together and pass the interrogation points. A typical menstruation cell and a schematic representation are shown in the succeeding text.

Equally tin be seen in Figure 6 , the flow cell is cone-shaped. By reducing the cantankerous-sectional area of the fluidic pathway, the final rate of fluid flow is increased and the cadre stream is focused tightly into the centre of the sheath. In analytic flow cytometers, information technology is the flow cell where the cells collaborate with the interrogation low-cal, which volition be discussed a bit after in this commodity.

In summary, the fluidic components of the period cytometry are responsible for moving the cells from the investigator's sample tube to the interrogation points. This is accomplished in menses cytometers using differential force per unit area between the sheath fluid and the sample fluid. This is similarly achieved within a pressure-, vacuum-, or a pump-based system. Utilizing the principles of laminar flow, the cells are hydrodynamically focused in the flow cell to be entrained in an organized mode as they pass through the interrogation point on their journey through the organization. To achieve a college event charge per unit at the sensing intercept, the differential pressure between the sheath and sample may be increased, but an excessive differential setting can result in more than coincident events, and poor quality data since the book of the sample cadre are besides increased and not all particles may be uniformly measured.

Finally, the laminar flow can be disrupted either physically, by aggregates and air bubbles, or chemically, past samples containing organic solvents and detergents. To prevent concrete disruption and artifacts that brand assay difficult, proper sample preparation, including filtration to remove clumps, is disquisitional, including a last resuspension in an acceptable sample buffer (typically phosphate-buffered saline containing some protein).

Read total chapter

URL:

https://www.sciencedirect.com/scientific discipline/article/pii/B9780123864567071021

Regulatory T Cell Therapy in Transplantation

Scott McEwen , Qizhi Tang , in Kidney Transplantation, Bioengineering and Regeneration, 2017

23.4.1.1 Treg Purification

None of the markers expressed on Tregs are unique to Tregs; rather, it is the combination of markers that uniquely identify Tregs (Table 23.ane ). When selecting markers to purify Tregs for clinical use, the practical issue of having access to skillful manufacturing practice (GMP)-class reagents poses a limit. Currently, antibodies confronting CD4, CD25, CD45RA, and CD127 are bachelor for GMP Treg manufacturing. What combination of markers can be used depends on the musical instrument of choice for the purification process, with magnetic activated cell sorters (MACS) or fluorescence activated cell sorting (FACS) being the two currently available instruments. MACS is available as a airtight GMP-compliant instrument but FACS remains to be a enquiry instrument. MACS has the reward of improve throughput past processing billions of cells in 1 batch as opposed to FACS selecting cells 1 at a time, although at very high speed. However, MACS is non as precise as FACS and so the resulting jail cell population is not as pure. This is a astringent limitation to MACS when the isolated Tregs are to exist expanded. Contaminating Tconv cells and CD8 ⁺ T cells proliferate much faster than Tregs, so the purity of cell preparation progressively declines in culture. FACS, by virtue of its precision and the ability to hands select cells based on multiple markers, tin can isolate highly pure Tregs. For instance, using the phenotype of CD4⁺CD127^lo/-CD25⁺, 1×10^vi highly pure Tregs tin can be isolated from 100 mL of blood on average. ¹³¹ These cells remain highly pure after expansion and show superior suppressive part in a humanized mouse model of transplantation. ¹³² Speed and the lack of a closed system are the major limitations to FACS. It currently takes eight–12 hours for a high speed FACS to process one unit of claret. Ideally, the best Treg purification procedure should incorporate the loftier throughput of MACS and the precision of FACS in one GMP-compliant instrument.

Tabular array 23.ane. Cellular Markers Relevant to Treg Immunotherapy

Cell marker	Alternative name	Function	Relevance to Treg therapy
CD3	TCR coreceptor complex	TCR signal transduction	Stimulation required for cell activation and proliferation
CD4		Interacts with MHC class Ii molecules to strengthen TCR binding	Identifies CD4⁺ lymphocyte subset
FoxP3	Forkhead box protein 3	Transcription factor, disquisitional regulator of Treg function	Identifies Tregs in CD4⁺ cells
CD25	IL-2 receptor a-chain	IL-2 receptor	Highly expressed in CD4⁺ Tregs
CD127	IL-seven receptor α-chain	IL-7 receptor	Negative Treg marker
CD28		Costimulation molecule necessary for T prison cell activation	Stimulation required for Treg proliferation
CTLA-4	Cytotoxic T lymphocyte antigen-4, CD152	Inhibitory signal that prevents T cell activation	Highly expressed in Tregs, important for suppressive function of Tregs
PD-i	Programmed expiry 1	Inhibitory bespeak that prevents T cell activation	Highly expressed in Tregs, marking of cell exhaustion
GITR	Tumor necrosis factor receptor (TNFR) superfamily member 18, glucocorticoid-induced TNFR-related protein	Cell signaling	Important mechanism of Treg suppression
GARP	Glycoprotein-A repetitions predominant	Cell signaling	Marker of activated Tregs, identifies thymic-derived Tregs
ICOS	Inducible T cell costimulator, CD278	Costimultory molecule on T cells	Stimulation leads to Treg proliferation and activation
LAG-3	Lymphocyte activation gene 3, CD223	CD4 homolog with MHC course Ii binding properties	Expressed on Tregs
LAP	Latency-associated peptide	Component of TGF-β latent complex	Identifies Treg subset with TGF-β-mediated function
CD45RA	Leukocyte common antigen (RA isoform)	Protein tyrosine phosphatase, receptor type, C	Identifies naïve cells
CD45RO	Leukocyte common antigen (RO isoform)	Protein tyrosine phosphatase, receptor type, C	Identifies experienced and memory cells
CD69	Transmembrane C-type lectin	Jail cell signaling	Marker of activated Tregs
CD62L	L-selectin	Lymphocyte prison cell adhesion molecule	Marker of activated Tregs
CD49b	Integrin VLA-iv α4β1α-chain	Cell adhesion and signaling	Expressed on Tregs
Helios		Dna binding protein	Marker of thymic-derived Tregs
CD103	Integrin αE	Cell adhesion and signaling	Identifies a Treg subset with increased suppressive part

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780128017340000230

Volume 1

Shiyu Cheng , ... Xingyu Jiang , in Encyclopedia of Biomedical Technology, 2019

Cell Assay

Cells are the bones units of organisms for life activities. Isolation and analysis of sure cells are quite powerful methods for biomedical research and applications. However, conventional approaches cannot manipulate cells precisely every bit the size of cells is quite small, only around 10 μm. In this field, microfluidic methods have excellent performance as the micro-fabricated structures allow meliorate spatial and temporal control of biological samples.

Circulating tumor cells (CTCs) are tumor cells that shed from primary lesions and broadcast in the bloodstream. CTCs are the main reason for tumor recurrence and cancer metastasis. And CTCs detection can be employed to cancer diagnosis and therapy monitoring. The main claiming for CTCs detection is that the abundance of CTCs in the bloodstream is very depression, compared with other blood cells. Many researchers have fabricated efforts to develop effective methods to isolate CTCs with high efficiency, either by physical strength difference or by the affinity difference.

We designed a label-free cell sorter for CTCs isolation ( Fig. 9A ). The jail cell sorter was composed of a double-spiral microchannel with i inlet and three outlets. When the claret sample flowed through the microchannel, dissimilar cells could be isolated due to their size difference. After separation, the pocket-sized blood cells flowed out from the inner outlet, while the large CTCs flowed out from the middle outlet. The recovery rate of tumor cells could exist 88.v%. When the summit of microchannel increased from 50 to 85 μm, the small cells flowed out from the centre outlet, and the large cells flowed out from the inner outlet (Fig. 9B). Spiked HeLa cells could exist separated successfully from the 20 × diluted blood sample using this cell sorter.

For the further analysis of CTCs, nosotros integrated an 8 μm filter on the outlet for cell enrichment (Fig. nineC). Afterwards separation, the CTCs were enriched by the filter. The capture efficiency of this cell sorter was determined to be 74.5 ± vi.1%, and the enrichment cistron reached 3.75 × x⁶. We used LAMP for CTCs analysis with CK-nineteen mRNA equally target nucleic acid. The results showed correlation with the assessment gotten from X-ray computed tomography (CT). Compared with FDA-approval CellSearch organisation, this method showed higher detection efficiency, especially for the epithelial mesenchymal transition (EMT) phenotype.

Microfluidic-based method has been widely used in biochemical analysis, both on molecule level and cell level. Compared with conventional methods, microfluidic-based methods are fourth dimension-saving, cost-saving and labor-saving. Besides, microfluidic-based devices have been broadly used in point-of-care testing area, every bit information technology is user-friendly to brand integrated platform by using microfluidic-based devices.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780128012383110293

New Sensing Technologies: Microtas/NEMS/MEMS

Hiroki Yasuga , ... Ryuji Kawano , in Reference Module in Biomedical Sciences, 2021

Introduction

Single-jail cell analysis is important for detailed analysis of the functions and prison cell-cell interactions, which are the smallest units of life. The report of prison cell function or structure started with microscopic ascertainment from the historic period of Leeuwenhoek. Subsequently, with the birth of the prison cell sorter and the mass production of microplates due to improvements in plastic molding engineering science, single-cell omics assay of various cells has come to be performed. In recent years, there has been an explosive increase in the number of research reports about mRNA sequencing analysis to encapsulate cells in a unmarried droplet i.due east. single-cell RNA sequencing (scRNA-seq) since the report by Macosko et al. (2015) and Klein et al. (2015) (Fig. 7). This method of using droplets is based on microfluidic devices, which was originally good at treatment microscale objects, and information technology is idea to have spread because of the successful integration with cell analysis, which is condign increasingly popular with the advent of next-generation sequencers (NGSs). In this affiliate, we will introduce a applied science that uses droplet based microfluidics to compartmentalize cells for the sensing of intracellular mRNA, while technologies for analyzing cells are increasingly being reported.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780128225486000467