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
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![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 <t>OP9</t> 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]](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7680/pmc13017680/pmc13017680__vkag012f6.jpg)