penicillin streptomycin  (Corning Life Sciences)

Journal: Oncotarget

Article Title: The IDO inhibitor 1-methyl tryptophan activates the aryl hydrocarbon receptor response in mesenchymal stromal cells

doi: 10.18632/oncotarget.20166

Figure Lengend Snippet: AHR nucleotransloaction in MSCs treated with 1MT and AHR agonists (A, B) MSCs were plated onto glass coverslips and allowed to adhere overnight. Media was aspirated and replaced with R10 or the indicated drug. Drugs were left on cells for 5h, after which cells were fixed and AHR was visualized via immunofluorescence; DAPI was used to visualize nuclei. Isotype-control was a non-specific murine-derived IgG. TCDD concentration was 10 nM. Concentrations of D-MT, L-MT, R-MT all at 1mM. These results (A, B) from an experiment with one MSC sample, which was replicated four times with independent MSC samples. All images were taken using a confocal microscope with the same exposure settings. (C) The bar graph represents the quantified results of nucleotranslocation, as observed via immunofluorsence. The Leica LASX software package was utilized to delimit regions of interest, defined by the DAPI-visualized nucleus. From these regions, the signal of Alexa488 was computed and normalized per μm 2 . These data are the cumulative average of three experiments using independent MSC samples, each with an average of twelve enumerations per high-power field. Statistical test performed was one-way ANOVA, P=0.003.

Article Snippet: Although culture-expanded in α-MEM, all subsequent tissue culture experimental work was performed in R10 (RPMI 1640 with L-glutamate plus 100 U/ml penicillin/streptomycin, and 10% fetal calf serum) (Corning International, Corning, NY).

Techniques: Immunofluorescence, Derivative Assay, Concentration Assay, Microscopy, Software

Journal: Oncotarget

Article Title: The IDO inhibitor 1-methyl tryptophan activates the aryl hydrocarbon receptor response in mesenchymal stromal cells

doi: 10.18632/oncotarget.20166

Figure Lengend Snippet: RNA-seq analysis of 1MT and TCDD treated MSCs (A) MSC samples (N=5) were cultured for 24h in vitro in the presence of R-MT (1 mM), TCDD (10 nM), or R10 vehicle (NoRx), and analyzed via mRNA-Seq. eat map displaying the union of all differentially-expressed genes (DEGs) found between control vehicle treated cells (NoRx) and TCDD treated cells or R-MT treated cells. DEGs were defined as +/−2-fold change and FDR

Article Snippet: Although culture-expanded in α-MEM, all subsequent tissue culture experimental work was performed in R10 (RPMI 1640 with L-glutamate plus 100 U/ml penicillin/streptomycin, and 10% fetal calf serum) (Corning International, Corning, NY).

Techniques: RNA Sequencing Assay, Cell Culture, In Vitro

Journal: Cell Reports

Article Title: Expression of RUNX1-ETO Rapidly Alters the Chromatin Landscape and Growth of Early Human Myeloid Precursor Cells

doi: 10.1016/j.celrep.2020.107691

Figure Lengend Snippet: RUNX1-ETO Induction Leads to Increased Survival and a Reversible Growth Arrest (A) RUNX1-ETO induction causes a reversible block in colony-forming ability. Top: diagram depicting the experimental strategy. EB cultures were treated with 3, 5, or 10 ng/mL Dox for 7 days, and suspension cells were subsequently plated in methylcellulose for CFU assays in the presence or the absence of Dox. Below: CFU assay of day 31 progenitors from treated EB cultures (at day 24 for 7 days), plated in methylcellulose in the presence (light gray) or absence (dark gray) of Dox. Data are from three independent biologic replicates using two clones. CFU assays were conducted in triplicate, with 3,000 cells plated per well. Error bars represent the standard error of the mean (SEM). Gray asterisk: multiple t test, statistical significance determined using the Holm-Sidak method, with alpha = 0.05. Each row was analyzed individually, without assuming a consistent SD. Black asterisk: two-way ANOVA, statistical significance determined using Dunnett’s multiple comparison test. (B) Induction of RUNX1-ETO at low levels (3 and 5 ng/mL Dox) enhances the survival of a subset of progenitor cells compared with uninduced cells, without increasing proliferative capacity. Left: schematic of the replating assays. EB cultures were treated at different stages of hematopoietic differentiation with 0, 3, 5, or 10 ng/mL Dox for 7 days. Floating progenitors were harvested and plated on Matrigel-coated wells at 2 × 10 5 cells/well in the corresponding Dox concentration and were serially passaged each week. Right: cell count of live cells × 10 5 during replating assays of hematopoietic progenitors from day 29 cultures previously treated with Dox for 7 days (from day 22), showing one representative of three biological replicates. Cell growth was measured at the indicated times. (C) RUNX1-ETO expressed at low levels allows survival of cells until day 87. Bright-field images of hematopoietic progenitors from replating assays at day 87 of differentiation that are uninduced (left) or treated with 3 ng/mL Dox from day 22 onward (right). Images are taken using the same magnification.

Article Snippet: At day 8, 20–30 EBs/well were transferred onto 6-well adherent plates pre-coated with Matrigel solution (IMDM with 1x Pen/Strep and 1:200 Corning® Matrigel® Growth Factor Reduced phenol red-free).

Techniques: Blocking Assay, Colony-forming Unit Assay, Clone Assay, Concentration Assay, Cell Counting