Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: ( A ) BM cells from tamoxifen-treated mice were cultured on high-attachment Primaria flasks or OP9 cell monolayers. Representative images show ZsGreen + cells at weeks 1, 3, and 8. ( B ) Workflow for culturing unsorted and sorted BM cell populations. Post-sort purity of ZsGreen + ECs is shown in the bottom left panel. All cells s were cultured (8 weeks) on OP9 cell monolayers supplemented with WT BM cells. Culture medium and floating cells were removed twice/week for 7 weeks. At the start of week 8, one final WT BM and medium supplementation was implemented prior to harvest at the end of week 8. Representative flow cytometry plots ( C ) and quantification ( D ) of CD45 + ZsGreen + cells from each of the 8 week cultures ( n = 5). ( E ) Floating/loosely adherent ZsGreen + cells from unsorted BM 8-week cell cultures were sorted and transplanted (5 × 10 4 , 2.5 × 10 4 , 1.25 × 10 4 , or 6.25 × 10 3 cells) into lethally irradiated (11 Gy) WT mice ( n = 2/group). Representative flow cytometry image ( F ) and quantification ( G ) of low-adherent cells harvested after 8 weeks of culture, showing that >95% (group average) of ZsGreen + low-adherent cells are CD45 + . These ZsGreen + CD45 + cells were sorted for transplantation. White blood cell (WBC) counts from five control mice (no irradiation or transplant) ( H ) and percent ZsGreen + , ZsGreen dim, and ZsGreen − cells ( I, J ) in blood of transplant recipients 10 months post-transplant ( n = 6). Dots represent individual mice. Data are shown as mean ± SD. ns, not significant by Student’s t -test.

Article Snippet: Cell line ( Mus musculus ) , OP9 cells , Dr. Nakano; same line deposited in ATCC , ATCC # CRL-2749; RRID: CVCL_4398 , Mouse bone marrow stromal cell line..

Techniques: Cell Culture, Flow Cytometry, Irradiation, Transplantation Assay, Control

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Related to . ( A ) Representative image (relates to ) showing the appearance of sorted ZsGreen + ECs after 4-week culture on OP9 monolayer. ( B ) Representative cytospin image of floating and low-adherent cells from ex vivo culture of BM cells from a tamoxifen-induced Cdh5- Cre ERT2 (PAC)/ZsGreen mouse.

Article Snippet: Cell line ( Mus musculus ) , OP9 cells , Dr. Nakano; same line deposited in ATCC , ATCC # CRL-2749; RRID: CVCL_4398 , Mouse bone marrow stromal cell line..

Techniques: Ex Vivo

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Percent EGFP + CD45 + cells in bone marrow (BM) and blood of tamoxifen-treated ( n = 6) or oil-treated ( n = 5) Col1a2-Cre ERT2 /mTmG mice ( A ) and tamoxifen-treated ( n = 4) or oil-treated ( n = 3) Col1a2-Cre ERT2 /ZsGreen mice ( B ). Cre-control mice ( n = 5 in A, and n = 2 in B). ( C ) Transplant experiment: sorted VE-Cadherin + CD45 − ZsGreen + /Col1a2 + cells from tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen mice are transplanted into 5-FU-conditioned WT recipients. Detection ( D ) and characterization ( E ) of ZsGreen + CD45 + cells in BM and blood of WT 5-FU-conditioned mice ( n = 5), 4 weeks post-transplant of VE-Cadherin + CD45⁻ZsGreen + /Col1a2 + cells. Control FU-conditioned WT mice ( n = 4) received no cell transplant ( D ). ( F ) Time course of ZsGreen + peripheral blood mononuclear cell (PBMC) detection in control (Cdh5-Cre + /ZsGreen + ) and Runx1 EC-KI (Cdh5-Cre + /ZsGreen + /Runx1-KI) mice ( n = 10 per group). Representative images ( G ) and quantification ( H ) of ZsGreen + cells from OP9 cell-supported cultures of BM cells from tamoxifen-treated Cdh5-Cre + /ZsGreen + ( n = 11) and Runx1 EC-KI mice ( n = 5). Representative flow cytometry plots ( I ) and quantification ( J ) of CD45 + ZsGreen + cells from OP9 cell-supported BM cell cultures ( n = 5/group). Dots represent individual mice. Data are shown as mean ± SD. **p < 0.01, ***p < 0.001 by Student’s t -test.

Article Snippet: Cell line ( Mus musculus ) , OP9 cells , Dr. Nakano; same line deposited in ATCC , ATCC # CRL-2749; RRID: CVCL_4398 , Mouse bone marrow stromal cell line..

Techniques: Control, Flow Cytometry

Journal: The Journal of Immunology Author Choice

Article Title: Suppression of pre-B cell colony formation by catecholamine oxidation

doi: 10.1093/jimmun/vkag012

Figure Lengend Snippet: Catecholamine mediated toxicity occurs in a feeder cell assay and B cell progenitors show a greater sensitivity to oxidative stress compared to other progenitor lineages. (A–B) Bone marrow B220 + cells were added to a layer of OP9 feeder cells. Cells were treated with isoproterenol and IL-7 was added to stimulate proliferation and differentiation. After 6 d of incubation, cells were harvested and B cell fractions were analyzed using flow cytometry [ n =4] (A) cell number and (B) frequency of B cell progenitor Fractions B, C, D. (C–D) Immune cell progenitor colony formation after treatment of murine bone marrow colony forming unit (CFU) assays with 10 µM isoproterenol. [ n =3]: (C) Granulocyte-monocyte progenitor (CFU-GM) colony counts (D) Early erythrocyte progenitor (burst forming unit-erythrocyte [BFU-E]) colony counts. (E–G) Murine bone marrow was seeded in media with cytokines and growth factors enabling the growth of multiple progenitor colonies (CFU-GM, BFU-E, CFU-E, and CFU-Pre-B). Cells were treated with 10 µM isoproterenol (ISO) or 5 µM menadione (MEN), and flow cytometry analysis was conducted after 6 d of incubation. [ n =6]: ( E ) Frequency of myeloid (CD45 + ,CD11b + ,CD19 − ) and erythroid (CD45 − ,CD71 + ) progenitors. ( F ) Frequency of B cell progenitors (CD45 + ,B220 + ,CD93 + ). ( G ) Representative flow cytometry plots showing B cell progenitors (B220 + CD93 + ), gated on CD45 + cells. B cell Fraction Definitions : All B cell fractions are CD45 + , B220 + , CD93 + ; (Fr.B: CD43 + , CD24 + ,BP1 − ), (Fr.C: CD43 + ,CD24 + ,BP1 + ), (Fr.D: CD43 − ,IgM − ,IgD − ) Statistical Analysis : Single pairwise comparisons: unpaired, two-tailed Student’s t test ( C, D ). Multiple pairwise comparisons: Two-way ANOVA with Dunnett’s test ( A, B, E, F ). [Data shown as mean ± standard deviation, ns= P > 0.05, *= P ≤ 0.05, **= P ≤ 0.01, ***= P ≤ 0.001, ****= P ≤ 0.0001]

Article Snippet: OP9 stromal cells ( ATCC ) were cultured in α-MEM ( Corning ) with 20% FBS + 1% P/S.

Techniques: Incubation, Flow Cytometry, Two Tailed Test, Standard Deviation