Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-1 was located on the upstream of miR-23a∼27a∼24-2 cluster and activated its expression during erythropoiesis. ( A ) Q-PCR analysis of the primary transcript of the miR-23a∼27a∼24-2 cluster in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( B ) Q-PCR analysis of the mature transcripts of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( C ) Northern blot analysis of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation. U6 snRNA was used as a loading control. ( D ) Q-PCR analysis of mature miR-27a and miR-24 expression in CD34+ HPCs undergoing day 4, 7, 11, 15 and 18 of E culture. ( E ) Representation of the human −603 bp miR-23a∼27a∼24 cluster promoter fragments. ( F ) ChIP-PCR and ChIP-qPCR analysis of the GATA-1 hit on the −557 site at the miR-23a∼27a∼24 cluster promoter in K562s. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01. ( G ) Immunoblot analysis of GATA-1 expression in K562s treated with siRNAs specific to GATA-1 (si_GATA-1) for 48 h or K562s transfected with a construct overexpressing GATA-1 (over_GATA-1) for 48 h, respectively. ( H ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (G). ( I ) Immunoblot analysis of GATA-1 expression in K562s undergoing erythroid differentiation. ( J ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( K ) Functional activity of GATA-1 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Northern Blot, Control, ChIP-qPCR, Standard Deviation, Western Blot, Transfection, Construct, Functional Assay, Activity Assay, Mutagenesis, Luciferase

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: MiR-27a and miR-24 promoted erythroid differentiation in CD34+ HPCs. ( A ) Monitoring of the GFP + population (left panel) and the CD235a stained GFP + fraction (medium panel) of Lenti-miRNA-transduced CD34+ HPCs on day 15 of E culture. The morphology (May-Grunwald Giemsa staining) of CD34+ HPCs derivatives on day 15 is shown in the right panel. A 400X magnification of a representative field is shown. ( B ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. Percentages of basophilic (Bas), polychromatophilic (Pol), orthochromatic (Ort) erythroblasts and erythrocytes (Ery) were determined by May-Grunwald/Giemsa staining of cytospin preparations. ( C ) Q-PCR analysis of gamma-globin mRNA expression in CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. ( D ) A comparison of the erythroid colony-forming capacity (CFU-E, BFU-E) of CD34+ HPCs transduced with Lenti-miRNAs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) FACS monitoring of CD34+ HPCs transduced with Zip-miRNA or Zip-GFP as described in (A). ( F ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Zip-27a, Zip-24 or Zip-GFP as described in (B). ( G ) Detection of gamma-globin mRNA level in CD34+ HPCs transduced with Zip-miRNA. ( H ) Colony-forming assay of CD34+ HPCs transduced with Zip-miRNA as described in (D).

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Staining, Transduction, Expressing, Comparison, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-2 was post-transcriptionally regulated by miR-27a and miR-24 during erythropoiesis. ( A ) A computer prediction of conserved and mutated binding sites within the 3′ UTR of GATA-2 mRNA for miR-27a and miR-24. ( B ) Relative luciferase activity of the indicated GATA-2 reporter constructs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( C ) Immunoblot analysis of GATA-2 in K562s transfected with scramble or miRNA mimics (miR-27a, miR-24) or inhibitors (Anti-27a, Anti-24). ( D ) Immunoblot analysis of GATA-2 in CD34+ HPCs transduced with Lenti-GFP control and lentivirus expressing miR-27a or miR-24 (Lenti-27a or Lenti-24). ( E, F ) ‘Rescue’ assays for miRNAs and GATA-2 during erythroid differentiation. Immunoblot analysis of GATA-2 in K562s treated with scramble or Anti-27a/24 (E) for 24 h. These cells were subsequently treated for another 24 h with control siRNAs or siRNAs specific to GATA-2 and were then treated with hemin treatment for 0, 48 and 72 h. (F) FACS analysis of K562s stained for CD71 and CD235a expression after 48 h of hemin induction as described earlier in the text.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, Transfection, Transduction, Control, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: The GATA switch regulated miR-27a and miR-24 expression. ( A ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1, GATA-2 and Pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( B ) Immunoblot analysis of GATA-1 and GATA-2 expression in K562s undergoing erythroid differentiation for 0, 24, 48 and 72 h. ( C ) Functional activity of GATA-1 and GATA-2 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( D ) Immunoblot analysis of GATA-2 in K562s treated with siRNAs specific to GATA-2 (si_GATA-2) for 48 h or K562s transfected with a construct overexpressing GATA-2 (over_GATA-2) for 48 h, respectively. ( E ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (D). ( F ) ChIP-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with control siRNAs or siRNAs specific to GATA-1/GATA-2 or K562s transfected with empty pcDNA3.1 vectors or pcDNA3.1_GATA-1/GATA-2. ( G ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in CD34+ HPCs of E culture. ( H ) Q-PCR analysis of Pri-27a-24 abundance in CD34+ HPCs of E culture (Top panel). Error bars represent the standard deviation obtained from three independent experiments. An immunoblot analysis of GATA-1 and GATA-2 expression in CD34+ HPCs of E culture (Bottom panel). ( I ) Q-PCR (left panel) and immunoblot (right panel) analysis of GATA-1 and GATA-2 expression in CD34+ HPCs transduced with lentivirus-expressing siRNAs against GATA-1 or control on day 11 of E culture. Error bars represent standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( J ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in CD34+ HPCs treated as described in (I). Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Standard Deviation, Western Blot, Functional Assay, Activity Assay, Mutagenesis, Luciferase, Transfection, Construct, Control, Transduction

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: A regulatory circuit involving GATA-1, GATA-2 and miR-27a/24 in erythropoiesis. ( A ) A schematic representation of the regulatory circuit comprised GATA-1, GATA-2, miR-27a and miR-24 in erythroid differentiation. ( B , C ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with scramble or miRNA mimics and inhibitors (B, r miR-27a and C, miR-24). ( D ) Q-PCR analysis of Pri-27a∼24 abundance in K562s treated as described in (B) and (C). Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Lenti-miR-27a/24 or Lenti-GFP control for 11 days. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( F ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Zip-miRNA or Zip-GFP for 11 days. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Transfection, Standard Deviation, Transduction, Control

Journal: iScience

Article Title: LncRNA IRAIN overcomes imatinib resistance in chronic myeloid leukemia via NF-κB/CD44 pathway inhibition

doi: 10.1016/j.isci.2024.109851

Figure Lengend Snippet: Low constitutive expression of IRAIN is associated with imatinib resistance, independent of BCR-ABL1, in CML (A) IRAIN expression levels were measured in healthy volunteers (N = 15) and patients with newly diagnosed CML (N = 17) and AML (N = 30) using qRT-PCR. GAPDH was used as a reference gene. (B) IRAIN levels were measured in CD34 + and CD34 − cells from patients with CML (N = 9) using qRT-PCR. Each line represents samples from the same patient. (C) IRAIN levels in imatinib-sensitive K562 cells and resistant K562/G01 cells were measured using qRT-PCR. Data are represented as mean ± SD of three technical replicates. (D) In K562 and MEG-01 cells, IRAIN was knocked down with siRNA against BCR-ABL1 (siBCR-ABL1) and the negative control siRNA (siNC). IRAIN and BCR-ABL1 transcripts were detected using qRT-PCR (Left), and BCR-ABL1 protein levels were detected using western blotting (Right) 48 h after transfection. The qRT-PCR data are represented as mean ± SEM of three independent experiments. Differences in expression between groups(A, B, C. and D) were calculated by unpaired t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Also, see and .

Article Snippet: The CD34 + cells were immunomagnetically enriched according to the instructions of the EasySep CD34 Positive Selection Kit (STEMCELL Technologies), and their purity was detected using a FITC-labeled anti-CD34 antibody by flow cytometry.

Techniques: Expressing, Quantitative RT-PCR, Negative Control, Western Blot, Transfection

Journal: iScience

Article Title: LncRNA IRAIN overcomes imatinib resistance in chronic myeloid leukemia via NF-κB/CD44 pathway inhibition

doi: 10.1016/j.isci.2024.109851

Figure Lengend Snippet: IRAIN overexpression enhanced the sensitivity to imatinib in purified CD34 + CML primary blasts ex vivo (A and B) Bone marrow mononuclear cells (BMMNCs) from two patients with CML (CML #4 and CML #5) were transfected with siRNA against IRAIN (siIRAIN) or the negative control siRNA (siNC). (A) The levels of IRAIN were detected using qRT-PCR after 48 h of transfection, with GAPDH used as a reference gene. Data are represented as mean ± SD of three technical replicates. (B) The transfected cells were treated with imatinib for 48 h, and imatinib-induced specific apoptosis levels were analyzed using flow cytometry through the Annexin V/PI dual staining. Data are represented as mean ± SEM of three biological replicates. (C‒E) RNA levels of IRAIN (C) were detected using qRT-PCR in BMMNCs obtained from patients with CML and transfected with the lentivirus expressing either IRAIN or the vector for 48 h. Data are represented as mean ± SD of three technical replicates. These transfected cells were treated with imatinib for another 48 h, and imatinib-induced specific apoptosis (D) was analyzed using flow cytometry through Annexin V/PI dual staining, and cell viabilities were detected using CCK8 assay. Data are represented as mean ± SEM of three biological replicates. Differences between groups (A, B, C, D and E) were calculated by unpaired Student’s t test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The CD34 + cells were immunomagnetically enriched according to the instructions of the EasySep CD34 Positive Selection Kit (STEMCELL Technologies), and their purity was detected using a FITC-labeled anti-CD34 antibody by flow cytometry.

Techniques: Over Expression, Purification, Ex Vivo, Transfection, Negative Control, Quantitative RT-PCR, Flow Cytometry, Staining, Expressing, Plasmid Preparation, CCK-8 Assay

Journal: Oncology letters

Article Title: Selective surface marker and miRNA profiles of CD34 + blast-derived microvesicles in chronic myelogenous leukemia.

doi: 10.3892/ol.2017.6336

Figure Lengend Snippet: Figure 1. CD123 expression is significantly increased in CD34+ cells of patients with CP‑CML and BC‑CML, compared with healthy donors. The gating strategy applied to determine the proportion of CD123+ cells in CD34+ subsets was: (A) P1, active cells; (B) isotype control; (C) Q2‑2, CD34+ blasts; (D) isotype control; (E) Q2‑8, CD34+ CD123+ blasts. (F) Proportion of CD123+ cells within CD34+ populations from healthy donors, and patients with CP‑CML and BC‑CML determined using multicolor flow cytometry. Each dot represents an individual tissue sample. P=0.105, N vs. CP‑CML; **P<0.01, CP‑CML vs. BC‑CML. CD, cluster of differentiation; CP, chronic‑phase; CML, chronic myelogenous leukemia; BC, blast crisis; N, healthy donors; SSC, side scatter; FSC, forward scatter; PE, phycoerythrin; Cy7, cyanine 7; Q, quadrant; Ig, immunoglobulin.

Article Snippet: CD34+ cells (>92% pure) were isolated using immunomagnetic separation by positive selection of cells (human CD34 microbead kit; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer's protocol.

Techniques: Expressing, Control, Flow Cytometry

Journal: Oncology letters

Article Title: Selective surface marker and miRNA profiles of CD34 + blast-derived microvesicles in chronic myelogenous leukemia.

doi: 10.3892/ol.2017.6336

Figure Lengend Snippet: Figure 2. CD123 expression is significantly increased in CD34+ blast‑derived MVs of patients with CP‑CML and BC‑CML compared with healthy donors. Flow cytometric analysis and representative cytograms of CD123+CD34+ blast‑derived MVs in patients with CML. (A) Fluorosphere beads of a known size were used as a standard to set the MV gate. P3 represents 1 µm beads. (B) P1 represents 3 µm beads. (C) P1 and P3 represent 3 µm and 1 µm beads, respectively. (D) Based on the calibrating beads above, P2 represents the MV gate. (E) IgG1‑FITC was set as the negative control for calcein‑AM+ MVs. (F) Events in the MV gate (P4) were analyzed to differentiate calcein‑AM+ MVs from the background signal. (G) Q2‑3, calcein‑AM+CD34+ MVs; (H) CD123 isotype control, Q2‑2, calcein‑AM+CD34+CD123‑ MVs. (I) Q2‑2, calcein‑AM+CD34+CD123+ MVs. (J) The proportion of CD123+ MV within calcein‑AM+CD34+ populations from N‑MV and patients with C‑MV and B‑MV was determined using multicolor flow cytometry. P=0.226, N‑MV vs. C‑MV; **P<0.01, C‑MV vs. B‑MV. CD, cluster of differentiation; CP, chronic‑phase; CML, chronic myelogenous leukemia; BC, blast crisis; MV, microvesicle; N‑MV, healthy; C‑MV, chronic‑phase CML; B‑MV, blast crisis CML; SSC, side scatter; FSC, forward scatter; PE, phycoerythrin; Cy7, cyanine 7; Q, quadrant; Ig, immunoglobulin; FITC, fluores cein isothiocyanate; Cal‑AM, calcein acetoxymethyl ester.

Article Snippet: CD34+ cells (>92% pure) were isolated using immunomagnetic separation by positive selection of cells (human CD34 microbead kit; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer's protocol.

Techniques: Expressing, Negative Control, Control, Flow Cytometry

Journal: Oncology letters

Article Title: Selective surface marker and miRNA profiles of CD34 + blast-derived microvesicles in chronic myelogenous leukemia.

doi: 10.3892/ol.2017.6336

Figure Lengend Snippet: Figure 5. Validation of miRNA expression levels using the reverse transcription‑quantitative polymerase chain reaction. Expression levels of miR‑627‑5p, miR‑483‑5p, miR‑638 and miR‑1290 in an independent set of primary CD34+ blasts and blast‑derived MVs were determined. miRNA expression in blasts was set as 1; the relative expression of hsa‑miR‑627‑5p, hsa‑miR‑483‑5p, hsa‑miR‑638 and hsa‑miR‑1290 in MVs was 2.09±0.35, 2.14±0.01, 5.59±0.69 and 3.92±0.46, respectively. Results are presented as the mean ± standard deviation. *P<0.05. miRNA, microRNA; CD, cluster of differentiation. MV, microvesicle; hsa, human.

Article Snippet: CD34+ cells (>92% pure) were isolated using immunomagnetic separation by positive selection of cells (human CD34 microbead kit; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer's protocol.

Techniques: Biomarker Discovery, Expressing, Polymerase Chain Reaction, Standard Deviation

Journal: Scientific Reports

Article Title: Smoothened Agonist Reduces Human Immunodeficiency Virus Type-1-Induced Blood-Brain Barrier Breakdown in Humanized Mice

doi: 10.1038/srep26876

Figure Lengend Snippet: ( a ) Experimental scheme for human CD34+ cell reconstitution, HIV infection, and time frame of observations. ( b) CD4+, CD8+ T cell percentages (N=8 per time point per group) and plasma viral load at 4 and 10 w.p.i. in HIV infected (N = 3 per time point per group). ***denotes P < 0.0001 as compared to CD4+ T cell percentages at baseline ( c) HIV protein p24 ELISA at 10 w.p.i. (N = 4 per group).

Article Snippet: Briefly, CD34 + cells were obtained from fetal liver and were enriched using immunomagnetic bead (CD34 + selection kit, #130-094-531 Miltenyi Biotech).

Techniques: Infection, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Comparative Mutational Profiling of Hematopoietic Progenitor Cells and Circulating Endothelial Cells (CECs) in Patients with Primary Myelofibrosis

doi: 10.3390/cells10102764

Figure Lengend Snippet: Molecular alterations on CD34 + -HSPCs and CECs. Mutated gene frequency and Molecular alterations discovered on CD34+-HSPCs ( A ) and CECs ( B ). On the top the frequency of mutated genes, while on the bottom the table with all the mutated genes in each patient.

Article Snippet: For CD34 + HSPC detection, 10 mL of PB was collected in EDTA (Ethylenediaminetetraacetic acid) tubes and examined within 6 h. HSPCs were selected using CD34+ immunomagnetic bead-column separation (magnetic-activated cell sorting (MACS) CD34 MicroBead Kit by Miltenyi biotech, 51429 Bergisch Gladbach, Germany).

Techniques:

Journal: Cells

Article Title: Comparative Mutational Profiling of Hematopoietic Progenitor Cells and Circulating Endothelial Cells (CECs) in Patients with Primary Myelofibrosis

doi: 10.3390/cells10102764

Figure Lengend Snippet: Comparative Somatic profiling of CD34 + -HSPCs and CECs in PMF patients. ( A ) Molecular profiles of both CEC and HSPC in patients with PMF. The molecular lesions found in the HSPC are in red, while in Green the ones discovered in the CEC. At the top of the table the clinical characteristics of patients, who successfully recovered CEC. ( B ) Mutated genes shared between HSPCs and CECs.

Article Snippet: For CD34 + HSPC detection, 10 mL of PB was collected in EDTA (Ethylenediaminetetraacetic acid) tubes and examined within 6 h. HSPCs were selected using CD34+ immunomagnetic bead-column separation (magnetic-activated cell sorting (MACS) CD34 MicroBead Kit by Miltenyi biotech, 51429 Bergisch Gladbach, Germany).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

doi: 10.1186/1479-5876-7-6

Figure Lengend Snippet: CD34 flow cytometry . Graphs show forward scatter versus fluorescence intensity. a) Unmarked control before CD34 purification with MACS columns, where 1.8% were CD34+. b) After CD34 purification with MACS columns, where 80.92% were CD34+. CD34+ cells are represented by pink points and CD34- cells are represented by blue points.

Article Snippet: Firstly, the immunomagnetically selected cells were incubated with the conjugated antibody anti-CD34-PerCP (BD Biosciences), in phosphate-buffered saline (PBS) at 4°C for 30 minutes, as recommended by the manufacturer.

Techniques: Flow Cytometry, Fluorescence, Purification

Journal: Journal of Translational Medicine

Article Title: Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

doi: 10.1186/1479-5876-7-6

Figure Lengend Snippet: Interaction between CD34+ stem cells and DMD myoblasts . a) after 1 hour (630×); b and c) after 24 hours (200×). arrow indicate syncytium. Microscope Zeiss Axiovert 200.

Article Snippet: Firstly, the immunomagnetically selected cells were incubated with the conjugated antibody anti-CD34-PerCP (BD Biosciences), in phosphate-buffered saline (PBS) at 4°C for 30 minutes, as recommended by the manufacturer.

Techniques: Microscopy

Journal: Journal of Translational Medicine

Article Title: Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

doi: 10.1186/1479-5876-7-6

Figure Lengend Snippet: Co-culture after 48 hours . Before the co-culture, stem cell nuclei were previously stained with Bisbenzimide H33342 (blue fluorescence). a) CD34+ stem cell nuclei with blue fluorescence, been (a) 200× and (a') 630×, respectively. b) Halogen light of the co-culture, showing the co-existence of both cells: fluctuant CD34+ stem cells and adherent myoblasts, been (b) 200× and (b') 630×, respectively. c) Pictures from panels a and b superposed, showing blue nuclei inside adherent cells (black arrows), been (c) 200× and (c') 630×, respectively. Microscope Zeiss Axiovert 200.

Article Snippet: Firstly, the immunomagnetically selected cells were incubated with the conjugated antibody anti-CD34-PerCP (BD Biosciences), in phosphate-buffered saline (PBS) at 4°C for 30 minutes, as recommended by the manufacturer.

Techniques: Co-Culture Assay, Staining, Fluorescence, Microscopy

Journal: Journal of Translational Medicine

Article Title: Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

doi: 10.1186/1479-5876-7-6

Figure Lengend Snippet: Dystrophin IF in culture cells . Anti-human dystrophin (N-terminal) FITC conjugated (green fluorescence) and nuclei dyed with Bisbenzimide H33342 (blue fluorescence). a) normal muscle cells, 200×; b) muscle cells of patient affected by DMD (dystrophin absent), 200×; c) Co-culture of stem cells CD34+ and muscle cells of patient affected by DMD, 200×. Microscope Zeiss Axiovert 200.

Article Snippet: Firstly, the immunomagnetically selected cells were incubated with the conjugated antibody anti-CD34-PerCP (BD Biosciences), in phosphate-buffered saline (PBS) at 4°C for 30 minutes, as recommended by the manufacturer.

Techniques: Fluorescence, Co-Culture Assay, Microscopy

Journal: Journal of Translational Medicine

Article Title: Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

doi: 10.1186/1479-5876-7-6

Figure Lengend Snippet: Dystrophin IF after 15 days in culture . Antibody anti-dystrophin N-terminal in green fluorescence. a) DMD muscle cells, after 15 days in culture, with nucleus dyed with Bisbenzimide H33342 (negative control); b) Co-culture after 15 days showing dystrophin expression and only the CD34+ stem cells' nuclei dyed with Bisbenzimide H33342. Microscope Zeiss Axiovert 200, 400×.

Article Snippet: Firstly, the immunomagnetically selected cells were incubated with the conjugated antibody anti-CD34-PerCP (BD Biosciences), in phosphate-buffered saline (PBS) at 4°C for 30 minutes, as recommended by the manufacturer.

Techniques: Fluorescence, Negative Control, Co-Culture Assay, Expressing, Microscopy

Journal: Journal of Translational Medicine

Article Title: Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

doi: 10.1186/1479-5876-7-6

Figure Lengend Snippet: CD34+ stem cells transdifferentiated in muscle cells in vitro . CD34+ stem cells that transdifferentiated in dystrophin producer muscle cells after 20 days in culture. Microscope Zeiss Axiovert 200.

Article Snippet: Firstly, the immunomagnetically selected cells were incubated with the conjugated antibody anti-CD34-PerCP (BD Biosciences), in phosphate-buffered saline (PBS) at 4°C for 30 minutes, as recommended by the manufacturer.

Techniques: In Vitro, Microscopy

Journal: Journal of Translational Medicine

Article Title: Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

doi: 10.1186/1479-5876-7-6

Figure Lengend Snippet: Western blot for dystrophin . WB analysis for dystrophin expression, after transdifferentiation of adherent cells obtained from prior CD34+ stem cells. Nc) normal control (human skeletal muscle). SC) CD34+ stem cells. aSC) adherent stem cells (prior CD34+).

Article Snippet: Firstly, the immunomagnetically selected cells were incubated with the conjugated antibody anti-CD34-PerCP (BD Biosciences), in phosphate-buffered saline (PBS) at 4°C for 30 minutes, as recommended by the manufacturer.

Techniques: Western Blot, Expressing