mouse methylcellulose complete media  (R&D Systems)

Journal: eLife

Article Title: Dissecting infant leukemia developmental origins with a hemogenic gastruloid model

doi: 10.7554/eLife.102324

Figure Lengend Snippet: ( A ) Immunostaining of whole individual haemGx at 144 hr showing the establishment of a vascular network by co-expression of CD31 and Flk1-GFP; nuclear staining is in DAPI (blue); scale bar: 100 µm. ( B ) Flow cytometry analysis of haemGx cells at 144 hr and 216 rhr stained for CD41, CD45, CD31, and CD34. ( C ) Quantification of flow cytometry analysis in ( B ) of CD41+ and CD45+ fractions co-expressing CD34 and CD31. Mixed effect analysis with a Ŝidàk test for multiple comparisons. ( D ) Colony-forming cell (CFC) assay of 216 hr-haemGx in multipotential methylcellulose-based medium. GEM: granulocyte-erythroid-monocyte; GM: granulocyte-monocyte; M: monocyte; E: erythroid. Please note that the medium does not include TPO and does not assess the presence of megakaryocytic progenitors. CFC frequency in 3 haemGx, n=3, mean ± SD. ( E ) Representative photographs of colonies quantified in C; scale bar = 2 mm. ( F ) Real-time quantitative (qPCR) analysis of expression of hemato-endothelial genes by timepoint. Relative gene expression fold change calculated by normalization to Ppia. Bars represent mean of three replicates and show individual data points; p-values by ANOVA across datapoints; post-hoc statistical significance between specific variables by Tukey’s test and shown by brackets.

Article Snippet: Disassembled gastruloids cells were plated on Mouse Methylcellulose Complete Media (R&D Systems).

Techniques: Immunostaining, Expressing, Staining, Flow Cytometry, Hematopoietic Colony Assay, Gene Expression

Journal: Frontiers in Immunology

Article Title: NR2F6 regulates stem cell hematopoiesis and myelopoiesis in mice

doi: 10.3389/fimmu.2024.1404805

Figure Lengend Snippet: Cytokines, CFUs, and the myeloid compartment in the bone marrow of Nr2f6 -deficient mice. (A) Quantification of G-CSF, M-CSF, and IL-1β concentrations (in pg/ml) in the bone marrow supernatant of wild-type ( Nr2f6 +/+ ) or Nr2f6 -deficient ( Nr2f6 -/- ) mice. (B, C) Quantification of the total CFU count (B) as well as specific colonies (C) , derived from the bone marrow in mouse methylcellulose complete media eight days post seeding of wild-type ( Nr2f6 +/+ ) or Nr2f6 -deficient ( Nr2f6 -/- ) mice. (D) Representative dot plots of bone marrow-derived CD45 + CD11b + , CD115 + , CD115 - , Ly6C hi and Ly6C mid-lo cell populations from wild-type ( Nr2f6 +/+ ) or Nr2f6 -deficient ( Nr2f6 -/- ) mice. (E-H) Quantification of bone marrow-derived CD11b + (E) , CD115 + (F) , Ly6C hi (G) and Ly6C mid-lo (H) cell populations within CD45 + cells from wild-type ( Nr2f6 +/+ ) or Nr2f6 -deficient ( Nr2f6 -/- ) mice. (I-J) Representative dot plots (I) and quantification (J) of bone marrow-derived Bst2 + B220 + pDCs within CD45 + cells from wild-type ( Nr2f6 +/+ ) or Nr2f6 -deficient ( Nr2f6 -/- ) mice. Representative data are shown as pooled experiments of two independent experiments, total n = 6/6 ( Nr2f6 +/+ )/( Nr2f6 -/- ) per genotype. Each dot represents the data of an individual mouse. Results are shown as median ± IQR with whiskers from min. to max. The Shapiro-Wilk test evaluated the normality of data. Outliers were excluded using ROUT (Q=1%) in GraphPad Prism. Asterisks indicate statistically significant differences between genotypes calculated using the nested t -test (B, C), Student’s t -test, or Mann-Whitney U test for non-parametric data. A p -value < 0.05 was considered statistically significant, *0.05, **0.01, (See also Supplementary Figure S5 ).

Article Snippet: According to the manufacturer’s protocol, CFU assays were conducted using the mouse methylcellulose complete media (R&D, HSC007).

Techniques: Derivative Assay, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: IRF2BP2 counteracts the ATF7/JDP2 AP-1 heterodimer to prevent inflammatory overactivation in acute myeloid leukemia (AML) cells

doi: 10.1093/nar/gkae437

Figure Lengend Snippet: IRF2BP2 depletion influences differentiation from mouse bone marrow stem and progenitor cells. ( A ) Western blot of components of the IRF2BP2 complex in cells isolated from mouse bone marrow. NUC = nucleoplasm, CHR = chromatin. ( B ) Representative bright field photography of colonies (here CFU-GM) obtained upon colony formation assays of myeloid progenitor cells with and without IRF2BP2 KD in methylcellulose medium. Additional examples are shown in . Scale = 500 μm. ( C ) Quantification of the area of colonies upon differentiation in methylcellulose. For each condition, 15 colonies were measured from two biological replicates. Significance was evaluated via a two-tailed unpaired Student's t -test. ( D ) Representative bright field microscopy photography of dendritic cell-shaped cells (arrows), seen in the IRF2BP2 KD cells compared to the control cells. Scale = 50 μm. ( E ) RT-qPCR analysis of collected cells after colony formation assays. Data represents the mean of three biological replicates. Significance was evaluated via a two-tailed paired Student's t -test. ( F ) FACS quantification of surface makers after 10 days of differentiation of Lin-negative bone marrow cells in liquid culture. Data represent the mean ± s.d. of at least three biological replicates. Significance was evaluated via a two-tailed unpaired Student's t -test. Example histograms are shown in . ( G ) GSEA of TCGA and our own RNA-Seq data regarding pathways involved in myeloid leukocyte migration and stem cell maintenance. n.s. = no significant; * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The cells were seeded in duplicates at 5 × 10 3 in 1 ml of mouse methylcellulose complete media without Epo (HSC008; R&D Systems) containing mIL-3, mIL-6 and mSCF (concentrations as described above), on 35 mm dishes and cultivated at 37°C and 5% CO 2 .

Techniques: Western Blot, Isolation, Two Tailed Test, Microscopy, Control, Quantitative RT-PCR, RNA Sequencing, Migration