clonogenic assay ppt

Sequencing data were processed to identify single‐nucleotide variants and copy‐number alterations (CNAs) which were subsequently used for testing of homologous recombination repair (HRR) deficiency.Enter your email address below and we will send you your usernameWhile we saw a decrease in OSRH‐2011/5 cell number in G1‐phase across all tested concentrations, we also saw a difference in the S and G2/M‐phase distribution after 48 hr.If the address matches an existing account you will receive an email with instructions to retrieve your usernameWe determined double‐stranded DNA breakage by seeding the primary osteosarcoma cell lines OSRH 2011/5 and OSKG and treating with prexasertib as indicated above.

(D) Clonogenic assay of sorted SP and MP cells treated with lenalidomide and thalidomide (10μM) is shown at day 14. Our study raises the intriguing possibility that lenalidomide could target SP cells with clonogenic potential, providing the framework for development of new treatment strategies targeting presumptive MM stem/tumor-initiating cells. 31.

This could also be observed in OSKG cells. This is underlined by γH2AX‐expression in this phase, leading to increased apoptosis as observed in both cell lines.Can't sign in? iv. Cell density during cloning. The clonogenic assay is the measurement of a drug's cytotoxic activity against a single cell population and is the most valid in vitro approach to predict the chemosensitivity of human cancers.

has received in the past consultant honoraria from Millennium Pharmaceuticals, Novartis Pharmaceuticals, Bristol-Myers Squibb, Merck & Co, Kosan Pharmaceuticals and Pharmion, licensing royalties from PharmaMar, and research funding from Amgen Pharmaceuticals, AVEO Pharma, EMD Serono, Sunesis, Gloucester, Genzyme, and Johnson & Johnson. Each point of the survival curve represents the mean surviving fraction from three independent experiments ± SD.
Other chemicals were purchased from Sigma-Aldrich, unless specifically mentioned.We thank Suzan Lazo-Kallanian for flow cytometry and Alexei Protopopov for FISH analysis.Conflict-of-interest disclosure: P.G.R. Cells may be washed in PBS at this stage, but it is important to keep all washes for Step 3.iii. ).The publication costs of this article were defrayed in part by page charge payment. Recurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations.

SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. Clonogenic survival assays enabled us to simultaneously analyze the proliferation of cells and the ability of single cells to produce progeny (forming a colony ≥50 cells). Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Cover the cells with trypsin–EDTA solution and incubate at 37°C until they become detached. was in the Speakers' Bureau (until 7/1/09) of Millennium and Celgene and received honoraria from Millennium and Celgene.

These results highlight the concentration‐dependent induction of DNA damage by prexasertib. 1. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account.At 24 and 48 hr after treatment initiation, both attached and detached cells were collected with EDTA/Trypsin (Biochrom GmbH), fixed in 4% paraformaldehyde solution (Merck KGaA) and permeabilized in ice‐cold 70% ethanol (Roth GmbH). ), and the Slovak R&D Agency grant VVCE-0001-07 (J.S. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.The online version of this article contains a data supplement.Contribution: J.J. designed and performed research, analyzed data, and wrote the paper; D. Cervi contributed to the writing of the paper; S.A., M.K.-A., J.F.D., and D. Cholujova performed research and analyzed data; S.K., M.L., S.B., M.O., S.-Y.K., and J.D.

and P.G.R.

Concentration of the toxic agent. At the end of the treatment, cells were washed with ice‐cold PBS and lysed with RIPA buffer. designed research and contributed to the writing of the paper.Thalidomide and lenalidomide (CC-5013, Revlimid) were provided by Celgene and dissolved in dimethyl sulfoxide (DMSO). We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Forgot your username?In sum, in both primary patient‐derived cell lines, γH2AX‐expression increased significantly in a time and concentration‐dependent manner. C.S.M. ii.