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: Isoproterenol alters the distribution of B cell progenitors at different stages of development in the CFU-Pre-B assay: Murine bone marrow was added to a CFU-Pre-B assay and treated with isoproterenol (ISO). After 4 and 6 d of incubation, cells were harvested and B cell progenitor fractions were analyzed with spectral flow cytometry. (A) cell number and (C) frequency of B cell progenitor Fractions B, C, and D after 4 d of incubation. (B) cell number and (D) frequency of B cell progenitor Fractions B, C, and D after 6 d of incubation. 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 : Multiple pairwise comparisons: Two-way ANOVA with Dunnett’s test; [Data shown as mean ± standard deviation, [ n =4], ns= P > 0.05, *= P ≤ 0.05, **= P ≤ 0.01, ***= P ≤ 0.001, ****= P ≤ 0.0001]

Article Snippet: One day after plating the OP9 cells, bone marrow was collected from a C57BL/6 mouse and magnetically sorted with CD45R (B220) MicroBeads ( Miltenyi Biotec ), positively selecting for B220 + bone marrow cells.

Techniques: Incubation, Flow Cytometry, Standard Deviation

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: Isoproterenol eliminates early pro-B cells with colony forming potential. (A–D) Murine bone marrow cells were stained with Cell Trace Violet (CTV), seeded into CFU-Pre-B media, and treated with 10 µM isoproterenol (ISO). After 3 d of incubation, B cells progenitors were analyzed with flow cytometry. [ n =5–6]: (A) Representative histograms of CTV staining in B cell progenitors (B220 + ,CD93 + ). Threshold for undivided cells was determined using non-proliferative bone marrow cells present in the CFU media. (B) Frequency of undivided cells within the B cell progenitor fractions. (C) Histograms showing CTV staining within Fraction B (Fr.B). Unstained Fraction B cells as a proportion of the total Fraction B cells. (D) Cell counts of B cell progenitor fractions after 3 d of incubation. (E) Proportion of Late-Pro B cells (Fr.C′) (CD24 hi ) within the Fraction C (CD24 + ) population, after different incubation periods of the CFU-Pre-B assay. [ n =3–6] B cell Fraction Definitions : All B cell fractions are CD45 + , B220 + , CD93 + ; (Fr.B: CD43 + , CD24 + ,BP1 − ), (Fr.C: CD43 + ,CD24 + ,BP1 + ), (Fr.C′: CD43 + ,CD24 hi , BP1 + ), (Fr.D: CD43 − ,IgM − ,IgD − ) Statistical Analysis : Single pairwise comparisons: unpaired, two-tailed Student’s t test ( C ). Multiple pairwise comparisons: Two-way ANOVA with Bonferroni’s test ( B, D ) or Sidak’s test ( E ); [Data shown as mean ± standard deviation, ns= P > 0.05, *= P ≤ 0.05, **= P ≤ 0.01, ***= P ≤ 0.001, ****= P ≤ 0.0001]

Article Snippet: One day after plating the OP9 cells, bone marrow was collected from a C57BL/6 mouse and magnetically sorted with CD45R (B220) MicroBeads ( Miltenyi Biotec ), positively selecting for B220 + bone marrow cells.

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

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: One day after plating the OP9 cells, bone marrow was collected from a C57BL/6 mouse and magnetically sorted with CD45R (B220) MicroBeads ( Miltenyi Biotec ), positively selecting for B220 + bone marrow cells.

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