igg1 fitc cd43 miltenyi biotec  (Miltenyi Biotec)

Journal: Development (Cambridge, England)

Article Title: NR4A1 and NR4A2 orphan nuclear receptors regulate endothelial-to-hematopoietic transition in mouse hematopoietic stem cell specification

doi: 10.1242/dev.201957

Figure Lengend Snippet: Nr4a genes are upregulated in the hemogenic endothelium during EHT. (A) Network schematic of the CLR-derived predictions at the 0.05 FDR for all HSC-specifying modules. Data taken from the StemSite portal ( http://daleystem.hms.harvard.edu/ ; ). Predicted transcriptional regulators are highlighted in red. Gene modules shown in gray squares. Names of ‘Hub’ genes are indicated. (B) Expression levels of Runx1 , Nr4a1 , Nr4a2 and Nr4a3 mRNA extracted from the StemSite portal . E9-YS (E9-CD41 + c-Kit + CD34 + YS cells), E11.5-AGM (E11.5 VE-cadherin + CD45 + AGM cells), E12.5-Pla (E12.5-CD45 + c-Kit + CD34 med placenta cells), E12.5-FL (E12.5 Lin − Sca-1 + c-Kit + VE-cadherin + Mac-1 low FL cells), E13.5-FL (E13.5 Lin − Sca-1 + c-Kit + CD150 + CD48 − FL cells), E14.5-FL (E14.5 Lin − Sca-1 + c-Kit + CD150 + CD48 − FL cells), adult WBM (Lineage − Sca-1 + c-Kit + CD150 + CD48 − adult BM cells), ESC (embryonic stem cells), EB (CD41 + c-Kit + cells isolated from day six mouse embryoid bodies) and ESC-HSC (CD41 bright CD45 − CD34 − cells) are shown . (C) Single-cell RNA-sequencing expression analysis of Runx1 , Nr4a1 and Nr4a2 mRNA. Data extracted from figure S3 of : E11-EC-AGM (E11-CD31 + VE-cadherin + CD41 − CD43 − CD45 − Ter119 − AGM cells); E11-T1 pre-HSC-AGM (E11 CD31 + CD45 − CD41 low c-Kit + CD201 high AGM cells); E11-T2 pre-HSC-AGM (CD31 + CD45 + c-Kit + CD201 high cells); E11-T2 CD41 low CD201 − AGM (E11 CD31 + CD45 + CD41 low CD201 − AGM cells); E12-FL-HSCs (E12-Lin − Sca-1 + Mac-1 low CD201 + FL cells); E14-FL-HSCs (E14-CD45 + CD150 + CD48 − CD201 + FL cells) and adult HSCs (CD150 + CD48 − Lineage − Sca-1 + c-Kit + cells). (D) Quantitative RT-PCR of Runx1 , Nr4a1 , Nr4a2 and Nr4a3 gene expression in VE-cadherin − CD45 − , VE-cadherin + CD45 − and VE-cadherin + CD45 + cells isolated from E11.5 AGMs in two independent experiments by pooling ∼50 AGMs/experiment. Flow cytometry gating strategies are shown in Fig. S1D . In B-D, gray boxes and red letters highlight hemogenic endothelium-related populations. Means and standard deviations are indicated. **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05 (Kruskal–Wallis tests). n =2-30. In B,C, each dot represents a different biological sample. In B, statistical differences were tested for each population compared to E11.5 AGM. In C, statistical differences were tested for each population compared to E11 EC-AGM. Source data are provided in Table S1 . CD150, SLAMF1; CD201, marker for EPCR (PROCR); Mac-1, ITGAM; Sca-1, LY6A; Ter119, LY76.

Article Snippet: For flow cytometry cell sorting or AGM analyses, dissociated cells were stained with one or more of these antibodies: CD45.2-V500 (104) (562130, BD Biosciences), CD41-PerCP-eFluor710 (eBioMWReg30, 46-0411-82, eBioscience), CD43-FITC (eBioR2/60, 11-0431-82, eBioscience), VE-cadherin (CD144)-PE (11D4.1, 138009, BD Biosciences), CD45-FITC (30-F11, 11-0451-82, eBioscience), c-Kit-APC-eFluor780 (2B8, 47-1171-82, eBioscience).

Techniques: Derivative Assay, Expressing, Isolation, RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Marker

Journal: International Journal of Molecular Sciences

Article Title: BAP31 Plays an Essential Role in Mouse B Cell Development via Regulation of BCR Signaling

doi: 10.3390/ijms25094962

Figure Lengend Snippet: BAP31−BCKO mice exhibit a defect in early B cell development. ( A ) Representative flow cytometry profiles of bone marrow (BM) from BAP31 fl/fl mice and BAP31 fl/fl CD19 Cre mice used to identify the percentages and numbers of prepro−B cells (IgM − B220 + CD43 + CD24 − CD19 − ), early pro−B cells (IgM + B220 + CD43 + ), pro−B cells (IgM − B220 + CD43 + CD24 + CD19 + ), pre−B cells (IgM + B220 + CD43 − ), immature B cells (IgM + B220 low ), and mature B cells (IgM + B220 hi ) ( n = 3). Numbers in the plots indicate percentages in each gate. ( B ) Aggregated data for the percentage and total number of different stages of bone marrow B cells. ( C ) B cell subpopulation in spleens of BAP31 fl/fl and BAP31 fl/fl CD19 Cre mice determined by means of flow cytometry ( n = 3). ( D ) Aggregated data for the percentage of subpopulation of splenic B cells. ( E ) FACS analysis of B cell subpopulations in the BM and spleen ( n = 3). ( F ) Aggregated data for the percentage of subpopulation of BM and splenic B cells. Each group included five mice, and measurements were repeated three times for each mouse; data are shown as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 compared with controls using unpaired Student t -tests. ns, no significant difference.

Article Snippet: For flow cytometry, PE-cyanine7-conjugated anti-B220 (clone: RA3-6B2), PE-conjugated anti-IgM (clone: II/41), anti-ICOSL (clone: HK5.3), anti-CD21 (clone: HB50), FITC-conjugated anti-CD43 (clone: eBIoR2/60), anti-CD3 (clone: 145-2C11), CD5 (clone: 53-7.3), anti-IgM (clone: II/41), Percp-cyanine5.5-conjugated anti-CD24 (clone: M1/69), and APC-conjugated anti-CD23 (clone: EBVCS2) were purchased from eBioscience( San Diego, CA, USA).

Techniques: Flow Cytometry