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TUNEL Assay

Introduction Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis. This assay is based on the incorporation of modified dUTPs by the enzyme TdT at the 3’-hydroxyl termini of fragmented DNA, a hallmark as well as the ultimate determinate of apoptosis. The modifications are fluorophores or haptens, including biotin of bromine which can be detected directly in the case of a fluorescently-modified nucleotide, or indirectly with streptavidin or antibodies, if biotin-dUTP or BrdUTP are used. Procedures: 1. Deparaffinize sections in 2 changes of xylene, and then hydrate with 2 changes of ethanol. 2. Pretreatment: Use the proteinase K digestion method. 3. Peroxidase Blocking. 4. Pre-incubate the sections in TdT Reaction Buffer. 5. TdT Reaction. 6. Detection. 7. Counterstain with Gill’s hematoxylin. 8. Clear in xylene. 9. Coverslip with xylene based mounting medium. Why Leading Biology? Working with us, you will get stability, and it means a reliable partne...

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Soft Agar Assay

Introduction The soft agar assay is a well-established method used to detect cell transformation and tumorigenicity. This assay requires that cells grow in an anchorage-independent manner, a hallmark of cancer cells, but not normal cells, moreover, growth in soft agar is strongly correlated to tumorigenicity in animals. Compare to the cells grown in 2D monolayers attached to plates, such 3D growth conditions more accurately reflect the natural environment of cancer cells and are crucial to be performed before animal studies. For this assay, cells (pretreated with carcinogens or carcinogen inhibitors) are cultured with appropriate controls in soft agar medium for 21-28 days. Following this incubation period, formed colonies can either be analyzed morphologically using cell stain and quantifying the number of colonies formed per well. Procedures: 1. Preparation of base agar. 2. Adding cell agar layer onto the base agar. 3. Formation of cell colonies. 4. Agar dissolve (homogeneous cell suspension formed). 5. Cell lysed and detected. Why Leading Biology? At Leading Biology, we custom protein puri...

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MTT Assay

Introduction MTT assay is an assay to detect cell viability which depends upon a mitochondrial dehydrogenase acting upon MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) to produce dark-blue formazan. The formazan is then solubilized, and the concentration determined by optical density at 570 nm. Tetrazolium dye reduction is dependent on NAD(P)H-dependent oxidoreductase enzymes largely in the cytosolic compartment of the cell, the reduction of MTT and other tetrazolium dyes depends on the cellular metabolic activity due to NAD(P)H flux. Cells with a low metabolism reduce very little MTT, while rapidly dividing cells exhibit high rates of MTT reduction. Thus, the MTT assay could be used for cell proliferation. Procedures: 1. Cell culture. 2. MTT incubation. 3. Dissolve the formazan by DMSO. 4. Detection by strip reader. 5. Data analysis. Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible. Working with us, you will get stability, and it means a reliable partner to help...

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Flow Cytometry-based Apoptosis Assay

Introduction Apoptosis, often called programmed cell death, is a carefully regulated process that is part of normal development and hemostasis. Because apoptosis is a dynamic event, and the time period during which cells exhibit apoptosis marker is variable and short, flow cytometry is an ideal technique for tracking cells through apoptosis. Early Apoptosis Flow Cytometry Detection: There are two separate dyes that could identify apoptotic and non-apoptotic cells: Annexin V binds to phosphatidylserine on the external membrane of apoptotic cells, and 7-AAD permeates and stains DNA of late-stage apoptotic and dead cells. Mid-Apoptosis Flow Cytometry Detection: During mid-apoptosis, we could measure Caspase activity using FLICA (fluorescent-labeled inhibitor of caspase) reagent, supplemented by a nuclear DNA stain 7-AAD, which evaluates membrane integrity and cell viability. Late Apoptosis Flow Cytometry Detection: At the late apoptosis stage, we use the TUNEL assay to detect the apoptosis-induced DNA fragmentation through a quantitative fluorescence assay. Terminal deoxynucleotidyl transferase (TdT) catalyzes the incorpor...

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Flow cytometry cell isolation and culture

Introduction Single-cell suspensions are required for all flow cytometry assays. Thus, peripheral blood cells or cells that grow in suspension are well suited for analysis by flow cytometry. Before flow cytometry analyzation start, the adherent cell lines should be separated into single-cell suspension, the suspension should remove cell clumps, dead cells, and debris which would affect the detection result. There are numerous protocols which could be used for cell separation, here are the procedures to prepare the tissue culture cells for flow cytometry: 1. Detach cells from the plate using trypsin or EDTA. 2. Collect the cells and dissociate and remove the clumps, dead cells, and debris. 3. Cell count and viability analysis. 4. Mixing cells with staining reagents, and incubation. 5. Centrifuge cells and wash them several times. 6. Resuspend the cells and ready to use. Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible. Working with us, you will get ...

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Fluorescence-activated Cell Sorting (FACS) Service

Introduction Fluorescence-activated cell sorting (FACS) is a derivative of flow cytometry which provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. The technique could provide fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. During FACS, the cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets, each droplet contains one cell. An electrical charging ring is placed just at the point where the stream breaks into droplets, the charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. Why Leading Biology? At Leading Biology, we custom protein purification design for ...

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Stable Cell Line Construction Service Using Lentivirus

Introduction Lentivirus can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector. There are many advantages of using lentivirus to generate stable cell lines: unlike short-term protein expression observed using transient transfection approaches, generating cell lines using lentiviral vectors enables long-term protein expression studies, additionally, engineered lentiviral transfer vector that is embedded with a matrix-attachment region (MARs) sequence may provide position-independent transgene expression. Compare to conventional stable cell line construction, transduction with lentivirus produces a much higher positive clone rate and the target always co-exists with the selection marker, thus reduce the cost, labor, and time spent selecting stable high expression clones. Procedures: 1. Construct the target gene into the lentiviral vector. 2. Generate lentivirus. 3. Transduce the interested cell line. 4. Select the stably transduced, high expression cells. 5. Validation of the genomic integration of genes via genomic PCR. 6. Validation of the high-expression clone ...

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Cell Cultivation Scale-up Process

Introduction Scale-up process involves the development of culture systems in stages from (small scale) laboratory to (large scale) industry. There are currently three main options available for scaling up adherent cell production, including keeping a 2D surface with some form of plate-based system; using microcarriers in stirred tank bioreactors and using some form of 3D technology where a sophisticated scaffold is used to hold the cell. To generate larger cell amounts, scale-out of the 2D approach has been suggested simply by multiplying culture dishes or by using multi-layered flasks. This method provides on the solution for both large autologous batches and small, allogenic batches containing a few billions of cells per batch, it would keeping a manufacturing lot size constant but multiplying the number of parallel unit operations. 3D culture is a potent approach to achieve the extensive cell number requirements. The bioreactor of 3D culture has a 3D scaffold structure based on a specific material such as nonwoven microfibers, a biopolymer, or a hydrogel, and the cells are entrapped in this fixed bed. This technology provides a good environment fo...

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Cell Line Construction Service

Introduction Cell culture and cell lines play an important role in studying physiological, pathophysiological and the differentiation processes of specific cells. A cell line is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space. The establishment of stable cell lines with high and reproducible protein expression is a crucial step in protein/antibody production, both for process optimization and for sustained productivity. There are many protocols available for stable cell line generation, here is the general protocol for cell line construction: 1. Probe design and construction. 2. Screening the most effective probes by QPCR with DOX induction. 3. Transduce the cell lines with effective probes. 4. Generation of stable polyclonal cell lines. 5. Quality Control. Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible. Working with us, you will get stability, and it means a reliable partner to help streamline your R&D proces...

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Primary Cell Isolation Service

Introduction Primary cells are cells taken directly from living tissue and established for growth in vitro. The source of primary cultures is excised animal tissue that is cultured either as an explant culture, suspension or as a monolayer and maintained in vitro. There are two types of isolated primary cells: 1. Adherent cells: cells that require attachment for growth are called adherent cells or anchorage dependent cells, these cells are mostly derived from tissues or organs where they are immobile and embedded in connective tissue. 2. Suspension cells: cells that do not require attachment for growth or do not attach to the surface of the culture vessels are called suspension cells or anchorage independent cells. These cells are derived from cells of the blood system. Procedures (all steps should be operated using sterile technique): 1. Mine/cut tissue. 2. Wash and add enzyme. 3. Incubate tissue specimen. 4. Disperse and wash cells. 5. Resuspend and measure cells. 6. Seed cells Why Leading Biology? At Leading Biology, we custom protein purification design for every single...

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