For stem cell assays, standardization is key to clinical. Calculate the volumes required to prepare a serial dilution for an assay. The cfu hill liquid medium kit has been developed specifically to support the culture and quantification of cfu hill colonies. Counting with colonyforming units requires culturing the microbes and counts only viable cells, in contrast.
Tuller fire assay method for gold determination is an old well known analytical method which is clearly described in iso. A second parent report form is suitable for presentation of the cfu assay. Mouse colonyforming unit cfu assays using methocult stemcell technologies inc. Different types of cytopenias are distinguished by the type of mature blood cell affected table 1.
This work was supported in part by national institutes of health grants hl55445 sa, hl51456 ha, ca096805 ha, ca 76226 ha and k18 par02069 ha. The cfu assay as an in vitro predictive tool in selecting an appropriate in vitro assay to assist in the prediction. The cfuhill liquid medium kit has been developed specifically to support the culture and quantification of cfuhill colonies. Oct 06, 2017 this video provides stepbystep guidelines for setting up hematopoietic colonyforming unit cfu assays using methylcellulosebased methocult medium. Automated imaging and analysis of hematopoietic cfu assays of. This makes the cfumk assay an excellent in vitro model for evaluating new drug candidates for their potential to cause thrombocytopenia in the clinic. Astm f2944 12 standard test method for automated colony. Optimisation of fire assay analyticaloptimisation of fire. A colonyforming unit cfu, cfu, cfu is a unit used in microbiology to estimate the number of viable bacteria or fungal cells in a sample. The pcr assay was a tripleplex reaction designed to.
This makes the cfu gm assay an excellent model for assessing the myelotoxic potential of new clinical compounds and predicting the level of clinical neutropenia they may cause. Faster and easier to use than any traditional cfu assay, even using automated colony counting. Fluorescence measurement was performed on spectramax gemini xs fluorometer molecular devices with a 485538 nm filter set and 530 nm cutoff. You must do this to find the dilution factor which yielded your cfu count. Cytoselect 96well hematopoietic colony forming cell assay. Calculates serial dilution using initial concentration and dilution factor or a concentration range. The number and functionality of myeloid progenitors can be accurately evaluated in the cfu gm assay, an in vitro colony forming cell cfc assay. Unique colonies that are formed in the 5day cfuhill colony assay are referred to as colonyforming unithill colonies or cfuhill colonies. Bouins fixative sigmaaldrich, ht102 protocol notes 1. Culture and quantification of cfu hill colonies using cfu. Development of a sensitive and specific biosensor assay to. The single cell gel electrophoresis assay scge, also known as comet assay is an uncomplicated and sensitive technique for the detection of dna damage at the level of the individual eukaryotic cell. Modifications to the original protocol were made to allow for the addition of the test compounds.
Hscs or hematopoietic progenitors known as colonyforming cells cfcs proliferate to form discrete colonies when cultured in a suitable 3d environment, such as methylcellulose supplemented with nutrients and cytokines. Sampling science read more about cell and biological sample cryopreservation. We measured the effect of 6 standard adriamycin, bcnu, dtic, melphalan, vinblastine, actinomycin d and 3 phase ii agents cisplatinum, vindesine, amsa on melanoma colonyforming units cfu in soft agar from biopsies of 50 patients with metastatic melanoma. Details colonyforming unit assay as a potency test for hematopoietic stemprogenitor cell products download download pdf.
However, manual colony counting is subjective, time consuming and results. An in vitro tool for predicting in vivo cytopenia tollfree t. While certain leukemic cfcs are morphologically distinct and can be visually distinguished from their normal counterparts, others look very similar to normal cfcs and can be confirmed as cancer cfcs by additional criteria in downstream assays e. Cfugm assay for evaluation of drug myelotoxic activity. In classical cfc assays, culture usually takes place in a 35 mm dish for 1421 days for the colonies to reach certain size 40 cellscolony for manual counting. Wp29840potency assays measuring engraftment potential hspcs. Serial dilution calculator and planner aat bioquest. In this paper, the creator will endeavor to characterize the part of rmm in pat and talk over the requisition of rmm to aseptic filling, biopharmaceutical upstream. Shop online for a wide selection of microbiologics ez cfu onestep microorganism preparations lyophilized microorganism preparations deliver less than 100cfu per 0. Stemvision automated colonyforming unit cfu assay reader. In the meantime, each gradient of cell sample was analyzed by ddpcr and qpcr.
The colonyforming unit or cfu assay is another test that can be used to measure not only the number but also the quality of stem cells in cb. The granulocytemacrophage colony forming unit gm cfu assay has been widely used to investigate the pathogenic mechanism of druginduced blood disorders and for screening the potential. Cfu assay, hematopoietic progenitor cell, potency test. Colonyforming cell cfc assays reachbio research labs. The following figures demonstrate typical results with the cytoselect 96well hematopoietic colony forming cell assay. The manual observerdependent assay the manual quantification of cell and cfu cultures based on observerdependent criteria or judgment is an extremely tedious and timeconsuming task and is significantly impacted by user bias. Revie article pen access shintani, pharm anal acta 20, 4. The assay involves taking an aliquot of hscs and culturing the cells in a semisolid medium containing the appropriate cytokines and. Melanoma cfu demonstrated marked heterogeneity in chemosensitivity to these 9 drugs. Viable is defined as the ability to multiply via binary fission under the controlled conditions. Mscs are the plastic adherent adult stemprogenitor cells from bone marrow originally referred to as fibroblastoid colony. Quantitation of drug sensitivity by human metastatic.
Table 5 dna per unit cfu titer dna content molecular. Following separation, cfu s were found as a sharp modal population of cells which was discrete from the modal populations of lymphocytes and other cell types. The efficiency with which they form colonies still remains an important assay for the quality of cell preparations. This video provides stepbystep guidelines for setting up hematopoietic colonyforming unit cfu assays using methylcellulosebased methocult medium. Velicity sedimentation separation demonstrated a heterogeneous cfuf sedimentation rate, with a modal sedimentation of 4. Nonsubjective, instrumentbased and quantitative assay system. Colonyforming unit cfu equivalent colorimetricabsorbance. This chapter describes 2 methods to assay the colony forming ability of mscs. Dna per unit cfu titer dna content molecular weight dna content dnacfu 102 x 106 x 90 x 402 x 106 426 x 106 72 x dnam1. The cfu assay takes 12 weeks to perform, but its important advantage is that it measures cell function, the ability of a stem cell to multiply and produce mature blood cells. Cfue is a stage of erythroid development between the bfue stage and the proerythroblast stage. Rapid, portable, and versatile detection of select agents. Im a bit leery about buying a whole machine to do this, but at some point if i want to beat tedious and cheap cfu counting im going to have to get real and buy something.
Using flow cytometry and a hillbased colony forming unit cfu assay, the present study indicated that higher levels of cd34 and vascular. In order to maintain consistency in data acquisition, pharmacological and drug discovery and development studies utilizing cell and colony. Population assay of biological indicator products lwi3108 rev 4 effective date. Cfus assay lara rossi, 2009 page 14 analysis of spleen colonies cfus assay at day 12. The assay is based on the ability of hematopoietic progenitors to proliferate and differentiate into colonies in a. Characterization of human bone marrow fibroblast colony. The cfc assay can be used in the cancer stem cell field to study leukemiccfc and cfus. It is this basic principle that allows the cfc assay to be standardized in m of atp rather than colony counts. Cfugemm colonyforming unitgranulocyte, erythrocyte, macrophage, megakaryocyte cfugm colonyforming unitgranulocyte, macrophage cfum colonyforming unitmacrophage. Several clinical studies have been conducted to determine which test better predicts whether and how quickly a patients bone marrow will be replenished after stem cell.
The assay is based on the ability of hematopoietic progenitors to proliferate and differentiate into colonies in a semisolid media in response to cytokine stimulation. Colonyforming unit cfu assay is a shortterm culture assay used for the detection of functionally active. Quantitation of drug sensitivity by human metastatic melanoma. Table 5 dna per unit cfu titer dna content molecular weight.
Lactose anhydrous nf dt sap code 20070652 general description lactose is a disaccharide obtained from the whey fraction of milk consisting of one glucose and one galactose moiety. Wp29840potency assays measuring engraftment potential. For human assays, the results of 7day and 14day cfu assays produced by stemvision can be printed in two different report formats. The granulocytemacrophage colony forming unit gmcfu assay has been widely used to investigate the pathogenic mechanism of druginduced blood disorders and for screening the potential. Refinement of the colonyforming unitmegakaryocyte cfu. Endothelial cell colony forming units derived from malignant breast. In this test, a very small sample of cb cells are grown in a culture dish containing growth factors. Absolute quantification of vibrio parahaemolyticus by.
Cfu assay correlations in univariate analysis between the indicated assay and clinical parameter hogge et al. A lab format report contains detailed information on the cfu assay for the laboratory or transplant physician. Start studying serial dilutions mastering microbiology lab homework. Refinement of the colonyforming unitmegakaryocyte cfumk. Mouse colonyforming unit cfu assays using methocult. Serial dilutions mastering microbiology lab homework. If you have previously done a yeast plate count, you will use this procedure with minor adjustments to assay the cfuml in a bacterial culture.
Cfumk assay for acute thrombocytopenia article in current protocols in toxicology chapter 20. Analysis of cfuf proliferative status by the thymidine suicide technique indicated that this cell. Generates a stepbystep protocol for planning serial dilutions. Ill have to check whether trypan blue works for bacteria. Soft agarose culture human tumour colony forming assay for drug.
The colony forming cell cfc assay, also referred to as the methylcellulose assay, is an in vitro assay used in the study of hematopoietic stem cells. This video provides stepbystep guidelines for setting up hematopoietic colony forming unit cfu assays using methylcellulosebased. Fluorescence measurement was performed on spectramax gemini xs fluorometer molecular devices with a 485538 nm filter set and 530 nm. Cfumk assay for acute thrombocytopenia request pdf. More ultras were mtbcpositive at lower cfu titres than xpert e. Shop online for a wide selection of microbiologics ezcfu onestep microorganism preparations lyophilized microorganism preparations deliver less than 100cfu per 0. Cfu we re enumerated and suspended in 200 l of sterile water.
Using the cfu assay to predict cytopenia cytopenia is defined as a reduced number of blood cells in the peripheral blood. Extract from used xpert mtbrif ultra cartridges is useful. Microbiologics ezcfu onestep microorganism preparations. Divide the cfu from the dilution 179 by the result from step 1 0. Counting with colonyforming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all. Sep 12, 20 the cfu assay is used to determine stem cell functionality, but it has not been approved for clinical use because of reproducibility issues between labs. If you have previously done a yeast plate count, you will use this procedure with minor adjustments to assay the cfu ml in a bacterial culture. Unique colonies that are formed in the 5day cfu hill colony assay are referred to as colonyforming unithill colonies or cfu hill colonies. It has since increased in popularity as a standard technique for evaluation of dna damagerepair. Rapid assay of airborne microorganisms and bioburden using several procedures. Rapid, portable, and versatile detection of select agents and. A collagenbased system megacultc, stemcell technologies, inc.
View of colonyforming unit assay as a potency test. Six days after 5fu injections, proceed to sacrifice. Cfue colony assay is designed to detect how many colonyformingunits of erythroid lineage there are in a hematopoietic tissue bone marrow, spleen, or fetal liver, which may be reflective of the organisms demand for oxygen delivery to the tissues or a hematopoietic disorder. Application note 50 years of colonyforming assays for. Main applications include elisa and other microplatebased experiments. The powder was swabbed and placed into 1 ml of biothreat alert bta buffer and tested in three ways. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Hematopoietic stem cells hscs are wellcharacterized, tissuespecific stem cells that are responsible for the lifelong maintenance of the hematopoietic system.
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