It involves removing a patients white blood cells from a. Sop for cfse proliferation assay fhhvtna0007 page 2 of 23 version 3. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Use of the intracellular fluorescent dye cfse to monitor lymphocyte migration and proliferation. Actg lab man dye dilution cfse proliferation 12 april 2004 lymphocyte proliferation using succinimidyl ester of carboxyfliorescein diacetate 1. Carboxyfluorescein succinimidyl ester cfse is a fluorescent cell staining dye. Quantitative analysis of cell proliferation by a dye dilution. For example the inhibitory effects on proliferation of antitnf antibodies can give an indication of the biological activity of the drug.
This sop describes how to perform a cfse proliferation assay in the fhcrc hvtn endpoint laboratory. Evaluation of t and b lymphocyte function in clinical. Assay your biologics or small molecules as single agents or. Make sure the cells are very well homogenized before plating. The proliferative response is usually measured with 3h. Cell proliferation assay by using microbeta 3hthymidine. Asymmetry of cell division in cfsebased lymphocyte.
Proliferation assay 3h thymidine incorporation analyzing lymphocyte proliferation can be a useful tool to determine immunomodulatory effects of biologics or other immunomodulatory drugs. The mean values of pi with cyst fluid antigens were 2. Mtt assay was performed according to the standard protocol. Importantly, asymmetry of protein distribution in lymphocyte division. The need for a reliable, sensitive and quantitative assay. Add 15 ml invitrogen ficollpaque plus to a 50 ml centrifuge tube and gently layer 20 ml diluted. The succinimidyl ester of carboxyfluorsecein diacetate 56 cfse is the best reagent currently available for the analysis of cellular proliferation. The sensitivity of an xtt assay is greatly improved by the usage of an intermediate electron carrier, pms nmethyl dibenzopyrazine methyl sulfate. Carefully remove lymphocyte layer and transfer to a new tube. Cfse is cell permeable and covalently couples, via its succinimidyl group, to intracellular molecules, notably, to intracellular. The unstimulated parent generation is indicated in blue. If the cells of one individual recognized others mhc molecules as foreign, they proliferate and divide.
The assay is based on the principles of measuring the membrane integrity that occur as a. The use of carboxyfluorescein diacetate succinimidyl ester. Principle, clinical applications and overview of the assay lymphocyte proliferation assay lpa measures the ability of lymphocytes placed in shortterm tissue culture to undergo a clonal proliferation when stimulated in vitro by a foreign molecule, antigen or mitogen. Cell based assays, cell viability, cell proliferation cro. Evaluation of t and b lymphocyte function in clinical practice using a flow cytometry based proliferation assay per maritsa,b, anncharlotte wikstroma,c, dusan popadicd, ola winqvista,b, sarah thunberga,e. Principle, clinical applications and overview of the assay lymphocyte proliferation assay lpa measures the ability of lymphocytes placed in shortterm tissue culture to undergo a clonal proliferation. Cell viability and proliferation assays sigmaaldrich.
T cell proliferation assay whole splenocytes or t cell. This method can be applied to immunological studies related to immunodeficiency, autoimmunity or infectious disease, where the lymphocyte proliferation. Celltrace cfse cell proliferation kit protocol thermo. Lymphocyte proliferation assay lymphocyte proliferation assay is used to determine lymphocyte activation and the cellmediated immune responses. Cell proliferation elisa, brdu colorimetric y version 17 content version. The results can be read on a multiwell scanning spectrophotometer elisa reader. Add 15 ml invitrogen ficollpaque plus to a 50 ml centrifuge tube and gently layer 20 ml diluted whole blood on top. The principle of t cell proliferation is that peripheral blood mononuclear cells pbmc in patients with chronic hbv infection are incubated with hbv epitope polypeptides and cytokines for 1. Then we explore the potential of multiplexed proliferation analysis.
The assay measures the lymphocyte proliferation after stimulating them with cyst fluid antigens. Lymphocyte proliferation assay lpa measures the ability of lymphocytes placed. There are a few main types of mlrs oneway, twoway, and threeway, and. In contrast to mtt, the cleavage product of xtt is soluble in water. We measure the division times of several cell lines using a dye dilution assay and compare these with values obtained from conventional proliferation assays and literature. Aug 26, 2010 this colorimetric assay measures the reduction of yellow 34,5dimethythiazol2yl2,5diphenyl tetrazolium bromide mtt by mitochondrial succinate dehydrogenase. I want to test a lymphocyte proliferation assay from collected blood of swine. Lymphocyte proliferation using succinimidyl ester of. Evaluation of a new lymphocyte proliferation assay based on cyclic. It allows for an overall comparison of the t cell driven antigenicity of any number of. A very common microbeta application is the cell proliferation assay performed with a method called thymidine incorporation.
The lymphocyte proliferation assay lpa is a test used to measure the ability of lymphocytes to proliferate in response to various stimuli such as candida, pokeweed and phytohaemagglutinin. Current in vitro testing methods that are being used. Pms helps drive xtt reduction and the formation of its formazan derivative. The amount of incorporated 3hthymidine is directly proportional to the number of cells present. This study was undertaken to address many of these variables and to determine whether lymphocyte proliferation assays correlate with clinical diagnoses. Carboxyfluorescein succinimidyl ester cfse is an effective and popular means to monitor lymphocyte division. A fluorescent azide, such as ifluor488, is then added. The introduction of t cell proliferation assay creative biolabs blog. The in vitro proliferation assay can be used to determine whether or not cells are triggered to divide after exposure to a specific stimulus, or to assess differences. Review and cite lymphocyte proliferation assay protocol, troubleshooting and other methodology information contact experts in lymphocyte. Comparison of whole blood and pbmc assays for tcell functional analysis.
A nonradioactive alternative to the 3hthymidine incorporation assay. Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. We introduced modifications to a standard proliferation protocol, and for the first time report antigen specific proliferation in blv infected animals with persistent. Apr 14, 2020 lymphocyte proliferation assay speaker. Flow cytometric analysis of t lymphocyte proliferation in.
Phytohemagglutinin, concanavalin a, pokeweed mitogen, candida antigen, and tetanus antigen are tested independently in lymphocyte culture. Cfse covalently labels longlived intracellular molecules with the. Studying the immune response may require an immune challenge, whether in a clinical immune study or in lab tests. Lymphocyte proliferation assay lpa assays for lymphocyte. Sop for cfse proliferation assay university of washington. August 2018 colorimetric immunoassay for the quantific ation of cell proliferation, based on the measurement of brdu incorporation during dna synthesis. Assay your biologics or small molecules as single agents or in combination in high throughput. Central memory t cells t cm cells express cd45ro, cc chemokine receptor type 7 ccr7, and lselectin cd62l.
Jun 23, 2014 porebski g, pecaricpetkovic t, grouxkeller m, bosak n, kawabata t, pichler wj 20 in vitro drug causality assessment in stevens johnson syndrome alternatives for lymphocyte proliferation assay. Porebski g, pecaricpetkovic t, grouxkeller m, bosak n, kawabata t, pichler wj 20 in vitro drug causality assessment in stevens johnson syndrome alternatives for lymphocyte proliferation assay. In edu staining, edu is incorporated into newly synthesized dna by cells within a sample. Cell proliferation using flow cytometry thermo fisher. Cells were then starved in serum free medium overnight.
A nonradioactive alternative to the 3hthymidine incorporation assay cat. The principle of t cell proliferation is that peripheral blood mononuclear. Oneway mlr is a cell proliferation assay where one population of lymphocytes responder cells is stimulated to proliferate by another population of lymphocytes stimulator cells, which have been. Horizons mixed lymphocyte reaction mlr assay allows for the rapid identification of agents that modulate apcmediated t cell activation. The reference ranges provided will be helpful in ascertaining the magnitude of the normal response. The tetrazolium salt xtt is cleaved to formazan by a complex cellular mechanism. Evaluation of the usefulness of lymphocyte proliferation. Edu 5ethynyl2deoxyuridine, a thymidine analog that is an alternative to brdu, is also used in dna proliferation assays that are simpler and faster than the brdu assay. The basic principle underlying the murine local lymph node assay llna is that sensitizers induce proliferation of lymphocytes in the lymph nodes draining the site of substance application. Cfse covalently labels longlived intracellular molecules with the fluorescent dye, carboxyfluorescein.
Lymphocyte proliferation assays are widely used to assess the cellmediated immunity. The invitrogen clickit edu and plus edu cell proliferation assays utilize the power of click chemistry and the modified nucleoside edu to provide a superior alternative to brdu staining for detecting and quantitating newly synthesized dna. A laboratory test that measures the ability of lymphocytes to replicate proliferate when exposed to an antigen. Optimization of lymphocyte proliferation assay for cells with high. The principle of the morphological method is to mix peripheral blood or separated single cells with certain phytohemagglutinin pha with a culture of 72h under 37. Flow cytometrybased proliferation assays have advantages in measuring cell division of different t lymphocyte. Lymphocytes are isolated from blood by lymphocyte separation medium 1. Mixed lymphocyte reaction mlr is a test used by pharmaceutical and biotech organizations to show the safety of a drug or implantable material. Mixed lymphocyte culture reaction mlc mlr principle. For example the inhibitory effects on proliferation. Mixed lymphocyte culture reaction mlc mlr principle t cells from one individual are mixed and cocultured with lymphocytes from a second individual. When good staining techniques are used, the cfse assay. Some investigators have claimed that lymphocyte proliferation assays are useful in the diagnosis of food hypersensitivity, despite the great variability in study designs and results reported.
Proliferation assay british society for immunology. A qualitative test of the immune system, the lymphocyte proliferation assay or mitogen assay is usually done in highly specialized research labs. Central memory t cells also have intermediate to high expression of cd44. Human t lymphocytes stained with the celltrace cfse cell proliferation kit and stimulated in culture for 5 days. Therefore, this lymphocyte proliferation is also called lymphocyte. Lymphocyte proliferation is then measured by the incorporation of 3hthymidine. The principle of t cell proliferation is that peripheral blood mononuclear cells pbmc in patients with chronic hbv infection are incubated with hbv epitope polypeptides and cytokines for 1 week. The colorimetric reduction of xtt by cellular enzymes xtt cell proliferation assay kit instruction manual catalog number 301011k assays store at 20oc this product is intended. Importantly, asymmetry of protein distribution in lymphocyte division is a basic biological feature of cell division with the degree of the asymmetry depending on various factors. Lymphocyte proliferation to mitogens is known to be affected by concomitant use of steroids, immunosuppressive agents, including cyclosporine, tacrolimus fk506, cellcept mycophenolate. Lymphocyte proliferation is defined as the process whereby lymphocytes begin to synthesize dna after crosslinking of their antigen receptor either following recognition of antigen or stimulation by a. Lymphocyte proliferation assay lpa measures the ability of lymphocytes placed in shortterm tissue culture to undergo a clonal proliferation when stimulated in vitro by a foreign molecule, antigen or mitogen. Dilute 10 ml whole blood in 10 ml pbs and mix well. Has it a rationale to use splenocytes from two congeneic mice 1 as responders from immunized mice, cfse labeled and 2 as apc from the second in t cell proliferation assay.
Lymphocyte proliferation assay many complex natural products contain immune modulating compounds that may rapidly alter immune cell function, migration, and activation status. Cell proliferation assay guide cell proliferation can be analyzed via flow cytometry. Primary immune deficiency abstract the golden standard for functional evaluation of immunodeficiencies is the incorporation of 3hthymidine in a proliferation assay stimulated with mitogens. The invitrogen clickit edu and plus edu cell proliferation assays utilize the power of click chemistry and the modified nucleoside edu to provide a superior alternative to brdu staining for detecting and. Evaluation of t and b lymphocyte function in clinical practice using a. Comparison of whole blood and pbmc assays for tcell. This sop describes how to perform a cfse proliferation assay. Lymphocyte proliferation an overview sciencedirect topics. When t cells are activated by antigenpresenting cells and cytokines. Proliferation assays on human t lymphocytes culture medium rpmi 1640 penicillin streptomycin 100 uml 100 gml lglutamin 4 mm sodium pyruvate 1 mm sigma s8636 non essential amino acids 1% sigma r71 rpmi vitamins 1% sigma r7256 mercaptoethanol 50 m sigma m6250 fetal calf serum 5% also needed. Testing natural products and lymphocyte proliferation. Mixed lymphocyte reaction mlr assay creative biolabs. Green cyanine dye assay the dye used for this cytotoxicity assay is a trademarked asymmetric green cyanine dye. Cell proliferation assays are widely used in cell biology for the study of growth factors, cytokines and media components, for the screening of cytotoxic agents and for lymphocyte activation.
Lymphocyte proliferation to mitogens is known to be affected by concomitant use of steroids, immunosuppressive agents, including. Lymphocyte proliferation assay lpa measures the ability of lymphocytes placed in shortterm tissue culture to undergo a clonal proliferation. During this period, formazon crystals will be formed at the. The mtt cell proliferation assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. T cells in patient peripheral blood will proliferate under the stimulation of hbv epitope peptide antigens. In physiological situations, contact between a lymphocyte and an antigenpresenting cell apc results in the formation of an immunological synapse. Proliferation assays on human t lymphocytes culture medium rpmi 1640 penicillin streptomycin 100 uml 100 gml lglutamin 4 mm sodium pyruvate 1 mm sigma s8636 non essential amino acids 1%. Mcf 10a cells were seeded at 1x104 cellswell in a 96well plate and incubated overnight. The introduction of t cell proliferation assay creative. Proliferation assays on human t cells baylor college of.
Mixed lymphocyte reaction is a test used by pharmaceutical and biotech organizations to show the safety of a drug or implantable material. The introduction of the cfse assay provided researches with the ability to derive those values, and, with sophisticated mathematical models, estimate values such as death rates, change in proliferation rates over time, and more 57. Lymphocyte proliferation is a fundamental characteristic of the response of lymphocytes to antigenic stimulation. Background principle of the xtt assay the xtt cell proliferation assay was first described in. It involves removing a patients white blood cells from a blood sample and exposing them to certain substances known as mitogens. Lymphocyte proliferation in response to these mitogens and antigens is determined by 3 hthymidine incorporation.
Lymphocyte proliferation assay definition aidsinfo. Under appropriate test conditions, this proliferation. Dec 20, 2019 the color can then be quantified using a simple colorimetric assay. This memory subpopulation is commonly found in the lymph nodes and in the peripheral circulation. Aug 17, 2017 there are two experimental types of t cell proliferation, morphological method, and radionuclide method. The mean values of proliferation index pi with cyst fluid antigens were 2. Evaluation of the mtt lymphocyte proliferation assay for the. If a product is capable of inducing cell proliferation directly, it can raise safety concerns. Monitoring t lymphocyte proliferation, especially in vivo, is essential for the evaluation of adaptive immune reactions. Thus, the basic assay doubles as a safety evaluation as well. It is commonly used as part of the fda clearance process. This video lecture explains the importance of mtt assay and the mechanism. It is often desirable to verify that a potent immunomodulatory product or compound does not, by itself, induce cell division proliferation.
The following protocol describes b lymphocyte proliferation in vitro induced by two commonly used stimulants, anti. This technique has been found useful in establishing the diagnosis of congenital t. A detailed protocol for the fascia procedure is available. In short, mixed lymphocyte reactions mlrs are cell proliferation assays. However, analysis of cfse proliferation assays is based on the premise that the label is halved in the two daughter cells.
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