cd34  (Miltenyi Biotec)

 
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    Name:
    CliniMACS CD34 Reagent System
    Description:
    The CliniMACS CD34 Reagent System is a medical device system that is designed for the in vitro enrichment of CD34 target cells from heterogeneous hematologic cell populations The process of CD34 cell selection results in simultaneous passive depletion of donor lymphocytes potentially obviating the need for immunosuppressive drugs to prevent GVHD
    Catalog Number:
    200-070-026
    Price:
    None
    Category:
    Cell therapy CliniMACS CD34 Reagent System FDA approved Health care professionals The FDA approved system CliniMACS CD34 Reagent System
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    Structured Review

    Miltenyi Biotec cd34
    CliniMACS CD34 Reagent System
    The CliniMACS CD34 Reagent System is a medical device system that is designed for the in vitro enrichment of CD34 target cells from heterogeneous hematologic cell populations The process of CD34 cell selection results in simultaneous passive depletion of donor lymphocytes potentially obviating the need for immunosuppressive drugs to prevent GVHD
    https://www.bioz.com/result/cd34/product/Miltenyi Biotec
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cd34 - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Reprogramming mechanisms influence the maturation of hematopoietic progenitors from human pluripotent stem cells"

    Article Title: Reprogramming mechanisms influence the maturation of hematopoietic progenitors from human pluripotent stem cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1124-6

    Donor-dependent variations in hematopoietic commitment capacity between human PSC lines. a Schematic diagram of serum- and feeder-free stepwise hematopoietic induction of human iPSCs and NT-ESCs. Flow cytometry analysis of committed hematopoietic progenitors (CD34+CD45+) and mature blood (CD34−CD45+) cells on day 17. b Representative images at different stages of hematopoietic differentiation. Scale bar, 100 μm. c, d Flow cytometry analysis of cells harvested on day 17 showing the frequencies of hematopoietic progenitor and mature blood cells. a p
    Figure Legend Snippet: Donor-dependent variations in hematopoietic commitment capacity between human PSC lines. a Schematic diagram of serum- and feeder-free stepwise hematopoietic induction of human iPSCs and NT-ESCs. Flow cytometry analysis of committed hematopoietic progenitors (CD34+CD45+) and mature blood (CD34−CD45+) cells on day 17. b Representative images at different stages of hematopoietic differentiation. Scale bar, 100 μm. c, d Flow cytometry analysis of cells harvested on day 17 showing the frequencies of hematopoietic progenitor and mature blood cells. a p

    Techniques Used: Flow Cytometry, Cytometry

    2) Product Images from "Variable Behavior of iPSCs Derived from CML Patients for Response to TKI and Hematopoietic Differentiation"

    Article Title: Variable Behavior of iPSCs Derived from CML Patients for Response to TKI and Hematopoietic Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071596

    Partial restoration of TKI-sensitivity of CD34 + hematopoietic progenitors derived from CML-iPSCs. Partial restoration of sensitivity to TKI of CD34 + hematopoietic progenitors derived from CML-iPSCs. Apoptosis in untreated versus imatinib cultures (5 µM, 24 h) was evaluated after annexin-V staining by FACS analysis, in CD34 + cells derived from CB-iPSC #11, CML-iPSCs #1.24 and #1.31.
    Figure Legend Snippet: Partial restoration of TKI-sensitivity of CD34 + hematopoietic progenitors derived from CML-iPSCs. Partial restoration of sensitivity to TKI of CD34 + hematopoietic progenitors derived from CML-iPSCs. Apoptosis in untreated versus imatinib cultures (5 µM, 24 h) was evaluated after annexin-V staining by FACS analysis, in CD34 + cells derived from CB-iPSC #11, CML-iPSCs #1.24 and #1.31.

    Techniques Used: Derivative Assay, Staining, FACS

    Characterization of iPSC clones. ( A ) Representative immunofluorescence of pluripotency markers in human iPSC clones derived from CD34 + CB cells (CB-iPSC #11) and CD34 + from CML first patient (CML-iPSCs #1.22, #1.24 and #1.31) and from CML second patient (#2.1 and #2.2), staining with anti-OCT4, anti-SOX2, anti-KLF4, anti-NANOG, anti-SSEA-4 and anti-TRA1-60. MEFs surrounding human iPSCs served as a negative control for immunofluorescence (magnification x100 or x200). ( B ) Representative alcian blue staining of histological sections of teratoma derived from human CB-iPSC #11 and CML-iPSC #1.31 encompassing tissues with all three germ layers (magnification x25 and x200).
    Figure Legend Snippet: Characterization of iPSC clones. ( A ) Representative immunofluorescence of pluripotency markers in human iPSC clones derived from CD34 + CB cells (CB-iPSC #11) and CD34 + from CML first patient (CML-iPSCs #1.22, #1.24 and #1.31) and from CML second patient (#2.1 and #2.2), staining with anti-OCT4, anti-SOX2, anti-KLF4, anti-NANOG, anti-SSEA-4 and anti-TRA1-60. MEFs surrounding human iPSCs served as a negative control for immunofluorescence (magnification x100 or x200). ( B ) Representative alcian blue staining of histological sections of teratoma derived from human CB-iPSC #11 and CML-iPSC #1.31 encompassing tissues with all three germ layers (magnification x25 and x200).

    Techniques Used: Clone Assay, Immunofluorescence, Derivative Assay, Staining, Negative Control

    Transgene independence of CML-iPSCs survival in presence of TKI. ( A ) PCR for the integrated vectors OSK 1 and MshP53 in 11 subclones of CML-iPSC #1.31 pretreated with CRE adenovirus. Generation of transgene-free subclone CML-iPSC #1.31i: excision of the 2 vectors. ( B ) Immunohistochemistry of pluripotency markers: OCT4, SOX2, KLF4, NANOG, SSEA-4 and TRA1-60 in human transgene-free iPSC subclones (after excision) derived from CD34 + from CML patient (#1.22 exc and #1.31 exc) ( C ) Dose-effect of TKI exposure (with imatinib (left panel) or ponatinib (right panel)) for 6 days on human excised CML-iPSCs (# 1.22, #1.31) and CB-iPSC (#11) subclones survival. iPSCs counts are conducted at day 6 and expressed as percentages relative to same iPSC clone without TKI. Mean ± SD of triplicate.
    Figure Legend Snippet: Transgene independence of CML-iPSCs survival in presence of TKI. ( A ) PCR for the integrated vectors OSK 1 and MshP53 in 11 subclones of CML-iPSC #1.31 pretreated with CRE adenovirus. Generation of transgene-free subclone CML-iPSC #1.31i: excision of the 2 vectors. ( B ) Immunohistochemistry of pluripotency markers: OCT4, SOX2, KLF4, NANOG, SSEA-4 and TRA1-60 in human transgene-free iPSC subclones (after excision) derived from CD34 + from CML patient (#1.22 exc and #1.31 exc) ( C ) Dose-effect of TKI exposure (with imatinib (left panel) or ponatinib (right panel)) for 6 days on human excised CML-iPSCs (# 1.22, #1.31) and CB-iPSC (#11) subclones survival. iPSCs counts are conducted at day 6 and expressed as percentages relative to same iPSC clone without TKI. Mean ± SD of triplicate.

    Techniques Used: Polymerase Chain Reaction, Immunohistochemistry, Derivative Assay

    Hematopoietic differentiation of CML-iPSCs. ( A ) Representative FACS analysis of CD45 + and CD34 + cells obtained from CB-iPSC #11, CML-iPSC #1.24 and CML-iPSC #1.31, after hematopoietic differentiation (at day 21), in non-adherent fraction. ( B ) Bar graphs showing average percentages of CD34 + , CD45 + and CD34 + /CD45 + cells obtained in non-adherent fractions at day 21 of hematopoietic differentiation (n = 5 independent experiments, mean ± SEM). ( C ) Western-blot analysis of total STAT3, phosphorylated STAT3 (p-STAT3) in Ph - iPSC (CB-iPSC #11 and CML-iPSC clones #1.22) and in Ph + iPSCs #1.24 and #1.31 in absence (−) or presence (+) of imatinib (20 µM) for 48 h. Murine embryonic stem cell extract (mES) in presence of LIF is used as positive control for STAT3 and pSTAT expression. ( D ) Bright field microscopy of colony forming units in methylcellulose medium (granulo-monocytic (CFU-GM) and erythroid (BFU-E)) obtained by hematopoietic cells derived from excised CB-iPSC #11 (upper panel) or Ph + CML-iPSC #1.31 (lower panel) (magnification x100). ( E ) FACS analysis of glycophorin A + and CD33 + cells obtained from Ph − iPSC #1.22, Ph + CML-iPSCs #1.24 and #1.31.
    Figure Legend Snippet: Hematopoietic differentiation of CML-iPSCs. ( A ) Representative FACS analysis of CD45 + and CD34 + cells obtained from CB-iPSC #11, CML-iPSC #1.24 and CML-iPSC #1.31, after hematopoietic differentiation (at day 21), in non-adherent fraction. ( B ) Bar graphs showing average percentages of CD34 + , CD45 + and CD34 + /CD45 + cells obtained in non-adherent fractions at day 21 of hematopoietic differentiation (n = 5 independent experiments, mean ± SEM). ( C ) Western-blot analysis of total STAT3, phosphorylated STAT3 (p-STAT3) in Ph - iPSC (CB-iPSC #11 and CML-iPSC clones #1.22) and in Ph + iPSCs #1.24 and #1.31 in absence (−) or presence (+) of imatinib (20 µM) for 48 h. Murine embryonic stem cell extract (mES) in presence of LIF is used as positive control for STAT3 and pSTAT expression. ( D ) Bright field microscopy of colony forming units in methylcellulose medium (granulo-monocytic (CFU-GM) and erythroid (BFU-E)) obtained by hematopoietic cells derived from excised CB-iPSC #11 (upper panel) or Ph + CML-iPSC #1.31 (lower panel) (magnification x100). ( E ) FACS analysis of glycophorin A + and CD33 + cells obtained from Ph − iPSC #1.22, Ph + CML-iPSCs #1.24 and #1.31.

    Techniques Used: FACS, Western Blot, Clone Assay, Positive Control, Expressing, Microscopy, Derivative Assay

    3) Product Images from "Effect of intravenous coadministration of human stroma cell lines on engraftment of long-term repopulating clonal myelodysplastic syndrome cells in immunodeficient mice"

    Article Title: Effect of intravenous coadministration of human stroma cell lines on engraftment of long-term repopulating clonal myelodysplastic syndrome cells in immunodeficient mice

    Journal: Blood Cancer Journal

    doi: 10.1038/bcj.2013.11

    Localization and colocalization of human HS27a stroma and hematopoietic cells in murine marrow (BM) and spleen. ( a ) Confocal microscopy showing colocalization of KG1a cells and HS27a stroma in fresh frozen sections of spleen (original magnification: × 40): HS27a cells are labeled with FITC-conjugated anti-human ICAM1 antibody. Red indicates human CD45+ KG1a cells, whereas blue shows nuclear staining with DAPI (both murine and human nuclei stain with DAPI). The right lower panel represents the merged picture. Superimposition of the FITC signals of the anti-ICAM1 antibody and the Alexa 647 signals of the CD45 antibody results in a yellow hue of signals. White arrows indicate examples of staining for colocalizing HS27a stroma and CD45+ cells. ( b ) Spleen sections (formalin fixed) stained with anti-human ICAM1 antibody (green) and anti-CD45 antibody (red), merged in the right panel (original magnification= × 40). The lower panels show isotype controls. ( c ) Immunohistochemical determination of the distribution of primary MDS cells (labeled with anti-human (h) CD34 and CD45 antibodies) and HS27a stroma (labeled with anti-human ICAM1 antibody) in the bone marrow and spleen of NSG mice. White arrows indicate identical coordinates on sequential sections (section distance=4 μm; orignal magnification= × 40). ( d ) Immunohistochemical staining of formalin-fixed spleen sections labeled with anti-human CD146 antibody (original magnification= × 40). Dark brown identifies 3,3′-diaminobenzidine chromagen linked to the CD146 antibody, identifying HS27a cells; blue represents counter staining with hematoxylin. Samples from two mice injected with primary CD45+ MDS cells from two different patients. Each figure represents one example of 2–4 similar experiments. ( e ) Flow cytometric analysis of bone marrow (BM) and spleen cells harvested from mice transplanted with human MDS marrow without coinjection of stroma (without stroma) or coinjected with unmodified HS5 or HS27a stroma. Cells positive for human ICAM1and CD146 (typical for HS27a stroma; see also Figure 4a and Supplementary Figure S4b ) were identified in marrow and spleen from mice injected with HS27a.
    Figure Legend Snippet: Localization and colocalization of human HS27a stroma and hematopoietic cells in murine marrow (BM) and spleen. ( a ) Confocal microscopy showing colocalization of KG1a cells and HS27a stroma in fresh frozen sections of spleen (original magnification: × 40): HS27a cells are labeled with FITC-conjugated anti-human ICAM1 antibody. Red indicates human CD45+ KG1a cells, whereas blue shows nuclear staining with DAPI (both murine and human nuclei stain with DAPI). The right lower panel represents the merged picture. Superimposition of the FITC signals of the anti-ICAM1 antibody and the Alexa 647 signals of the CD45 antibody results in a yellow hue of signals. White arrows indicate examples of staining for colocalizing HS27a stroma and CD45+ cells. ( b ) Spleen sections (formalin fixed) stained with anti-human ICAM1 antibody (green) and anti-CD45 antibody (red), merged in the right panel (original magnification= × 40). The lower panels show isotype controls. ( c ) Immunohistochemical determination of the distribution of primary MDS cells (labeled with anti-human (h) CD34 and CD45 antibodies) and HS27a stroma (labeled with anti-human ICAM1 antibody) in the bone marrow and spleen of NSG mice. White arrows indicate identical coordinates on sequential sections (section distance=4 μm; orignal magnification= × 40). ( d ) Immunohistochemical staining of formalin-fixed spleen sections labeled with anti-human CD146 antibody (original magnification= × 40). Dark brown identifies 3,3′-diaminobenzidine chromagen linked to the CD146 antibody, identifying HS27a cells; blue represents counter staining with hematoxylin. Samples from two mice injected with primary CD45+ MDS cells from two different patients. Each figure represents one example of 2–4 similar experiments. ( e ) Flow cytometric analysis of bone marrow (BM) and spleen cells harvested from mice transplanted with human MDS marrow without coinjection of stroma (without stroma) or coinjected with unmodified HS5 or HS27a stroma. Cells positive for human ICAM1and CD146 (typical for HS27a stroma; see also Figure 4a and Supplementary Figure S4b ) were identified in marrow and spleen from mice injected with HS27a.

    Techniques Used: Confocal Microscopy, Labeling, Staining, Immunohistochemistry, Mouse Assay, Injection, Flow Cytometry

    Effect of CD146 expression in stroma on BrdU uptake by cocultured CD34+ hematopoietic cells in vitro , and on engraftment of CD34+ MDS marrow cells in NSG mice in vivo . ( a ) Flow cytometric analysis showed prominent expression of CD146 on HS27a, but not on HS5 stroma. Knockdown (KD) of CD146 in HS27a cells using four different siRNAs (no. 1–4 in comparison with a scrambled siRNA) reduced CD146 expression to levels comparable to those in HS5 cells. Conversely, overexpression of CD146 (over-CD146) in HS5 cells using a lentiviral construct (pLOC vector, over-CD146, in comparison to a scrambled vector (SCR)) increased CD146 expression to levels comparable to those in unmodified HS27a cells (see also Supplementary Figure S4b ; upper row). ( b ) BrdU uptake by hematopoietic cells after coculture with either unmodified HS27a stroma wild type (WT), HS27a cells with KD of CD146 (146 KD), or HS5 cells overexpressing CD146 (over-CD146), in comparison with unmodified (WT) HS5 cells. BrdU uptake was highest in KG1a cells, followed by MDS-derived CD34+ cells and CD34+ cells from healthy donors. Results of coculture with HS27a cells with KD of CD146 approached those with unmodified HS5 cells, whereas, conversely, BrdU uptake in coculture with CD146 overexpressing HS5 cells did not differ significantly from that in coculture with unmodified HS27a cells (Student's t -test; mean±s.e.m. of three experiments ). ( c ) Engraftment of CD45+ marrow cells from two patients with RAEB-2 and RAEB-1, respectively, coinjected with unmodified HS5 stroma (HS5), unmodified HS27a stroma (HS27a) or HS5 cells overexpressing CD146 (HS5-CD146), in marrow and spleen of NSG mice, determined at 5–7 weeks after transplantation. The table shows, in addition, the proportions of human clonal and non-clonal CD34+cells (from patient 23) in mouse marrows and spleens after coinjection with HS27a and HS5-CD146 cells (day 35), respectively. Additional data on FISH and flow cytometric analysis, as well as immunohistochemical analysis of ICAM1 and CD146 expression are shown in Supplementary Figure S4 . RAEB-1 or 2, refractory anemia with excess blasts 1 or 2, respectively; NBM, normal bone marrow.
    Figure Legend Snippet: Effect of CD146 expression in stroma on BrdU uptake by cocultured CD34+ hematopoietic cells in vitro , and on engraftment of CD34+ MDS marrow cells in NSG mice in vivo . ( a ) Flow cytometric analysis showed prominent expression of CD146 on HS27a, but not on HS5 stroma. Knockdown (KD) of CD146 in HS27a cells using four different siRNAs (no. 1–4 in comparison with a scrambled siRNA) reduced CD146 expression to levels comparable to those in HS5 cells. Conversely, overexpression of CD146 (over-CD146) in HS5 cells using a lentiviral construct (pLOC vector, over-CD146, in comparison to a scrambled vector (SCR)) increased CD146 expression to levels comparable to those in unmodified HS27a cells (see also Supplementary Figure S4b ; upper row). ( b ) BrdU uptake by hematopoietic cells after coculture with either unmodified HS27a stroma wild type (WT), HS27a cells with KD of CD146 (146 KD), or HS5 cells overexpressing CD146 (over-CD146), in comparison with unmodified (WT) HS5 cells. BrdU uptake was highest in KG1a cells, followed by MDS-derived CD34+ cells and CD34+ cells from healthy donors. Results of coculture with HS27a cells with KD of CD146 approached those with unmodified HS5 cells, whereas, conversely, BrdU uptake in coculture with CD146 overexpressing HS5 cells did not differ significantly from that in coculture with unmodified HS27a cells (Student's t -test; mean±s.e.m. of three experiments ). ( c ) Engraftment of CD45+ marrow cells from two patients with RAEB-2 and RAEB-1, respectively, coinjected with unmodified HS5 stroma (HS5), unmodified HS27a stroma (HS27a) or HS5 cells overexpressing CD146 (HS5-CD146), in marrow and spleen of NSG mice, determined at 5–7 weeks after transplantation. The table shows, in addition, the proportions of human clonal and non-clonal CD34+cells (from patient 23) in mouse marrows and spleens after coinjection with HS27a and HS5-CD146 cells (day 35), respectively. Additional data on FISH and flow cytometric analysis, as well as immunohistochemical analysis of ICAM1 and CD146 expression are shown in Supplementary Figure S4 . RAEB-1 or 2, refractory anemia with excess blasts 1 or 2, respectively; NBM, normal bone marrow.

    Techniques Used: Expressing, In Vitro, Mouse Assay, In Vivo, Flow Cytometry, Over Expression, Construct, Plasmid Preparation, Derivative Assay, Transplantation Assay, Fluorescence In Situ Hybridization, Immunohistochemistry

    Human MDS marrow cells in spleen and bone marrow of mice coinjected with MDS marrow and HS27a or HS5 stroma cells, respectively. ( a and b ) Spleen and marrow cells were harvested at weeks 12 or 13 after transplantation and analyzed by flow cytometry. The percentages of cells in the respective quadrants are indicated. The flow analysis shows results obtained with cells from patients 14 and 18, respectively ( Table 1 ). ( c ) Immunohistochemical staining of spleen and femur from NSG mouse engrafted with MDS marrow. Labeling with anti-human CD34 and CD45 antibodies appears as brown signals. ( d ) Summary of engraftment of human HPC in four experiments comparing the effects of HS5 and HS27a (box plot: lowest, 1st quartile, median, 3rd quartile, highest value). BM, bone marrow (Comparison by Student's t -test).
    Figure Legend Snippet: Human MDS marrow cells in spleen and bone marrow of mice coinjected with MDS marrow and HS27a or HS5 stroma cells, respectively. ( a and b ) Spleen and marrow cells were harvested at weeks 12 or 13 after transplantation and analyzed by flow cytometry. The percentages of cells in the respective quadrants are indicated. The flow analysis shows results obtained with cells from patients 14 and 18, respectively ( Table 1 ). ( c ) Immunohistochemical staining of spleen and femur from NSG mouse engrafted with MDS marrow. Labeling with anti-human CD34 and CD45 antibodies appears as brown signals. ( d ) Summary of engraftment of human HPC in four experiments comparing the effects of HS5 and HS27a (box plot: lowest, 1st quartile, median, 3rd quartile, highest value). BM, bone marrow (Comparison by Student's t -test).

    Techniques Used: Mouse Assay, Transplantation Assay, Flow Cytometry, Cytometry, Immunohistochemistry, Staining, Labeling

    Extent of engraftment and clonality of human MDS marrow cells in secondary NSG recipients: analysis by flow cytometry and FISH. ( a ) FISH detection of del 5q (two green and one red signal identify cells with 5q-), trisomy 8 (three red signals indentify cells with +8), del 20q (two red and one green signal indentify cells with 20q-) and a CBFB/MYH11 dual fusion translocation (one green and one red merging indentifies a cell with the CBFB/MYH11 translocation). ( b ) Flow cytometric profiles of marrow cells recovered from secondary NSG recipients 13 weeks after transplantation of CD34+ human cells that had been harvested from the primary recipients 12 weeks after the original transplant. Human CD45+ and CD34+ cells are present. The percentages of cells in the respective quadrants are indicated (human cells were derived originally from patients 14 and 18, respectively; Table 1 ). ( c ) FISH shows del 5q (two green and one red signal identifying cells with 5q-) in the spleen of the secondary recipient. BM, bone marrow.
    Figure Legend Snippet: Extent of engraftment and clonality of human MDS marrow cells in secondary NSG recipients: analysis by flow cytometry and FISH. ( a ) FISH detection of del 5q (two green and one red signal identify cells with 5q-), trisomy 8 (three red signals indentify cells with +8), del 20q (two red and one green signal indentify cells with 20q-) and a CBFB/MYH11 dual fusion translocation (one green and one red merging indentifies a cell with the CBFB/MYH11 translocation). ( b ) Flow cytometric profiles of marrow cells recovered from secondary NSG recipients 13 weeks after transplantation of CD34+ human cells that had been harvested from the primary recipients 12 weeks after the original transplant. Human CD45+ and CD34+ cells are present. The percentages of cells in the respective quadrants are indicated (human cells were derived originally from patients 14 and 18, respectively; Table 1 ). ( c ) FISH shows del 5q (two green and one red signal identifying cells with 5q-) in the spleen of the secondary recipient. BM, bone marrow.

    Techniques Used: Flow Cytometry, Cytometry, Fluorescence In Situ Hybridization, Translocation Assay, Transplantation Assay, Derivative Assay

    4) Product Images from "Antisense STAT3 inhibitor decreases viability of myelodysplastic and leukemic stem cells"

    Article Title: Antisense STAT3 inhibitor decreases viability of myelodysplastic and leukemic stem cells

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI120156

    STAT3 downregulation by AZD9150 leads to downregulation of MCL1. ( A ) Positive correlation between MCL1 and STAT3 expression is seen in 183 MDS CD34 + samples. MCL1 protein downregulation is seen after treatment of leukemia cells with AZD9150. ( B ) Western blot showing downregulation of MCL1, phospho-STAT3, and total STAT3 in AZD9150-treated CMK cells compared with the control. ( C ) STAT3 ChIP for MCL1 promoter region in CMK cells shows enrichment when compared with IgG control ( P = 0.03, n = 2). * P
    Figure Legend Snippet: STAT3 downregulation by AZD9150 leads to downregulation of MCL1. ( A ) Positive correlation between MCL1 and STAT3 expression is seen in 183 MDS CD34 + samples. MCL1 protein downregulation is seen after treatment of leukemia cells with AZD9150. ( B ) Western blot showing downregulation of MCL1, phospho-STAT3, and total STAT3 in AZD9150-treated CMK cells compared with the control. ( C ) STAT3 ChIP for MCL1 promoter region in CMK cells shows enrichment when compared with IgG control ( P = 0.03, n = 2). * P

    Techniques Used: Expressing, Western Blot, Chromatin Immunoprecipitation

    Important functional pathways are dysregulated in MDS CD34 + samples with high expression of STAT3. ( A ) Gene expression profiles from samples with low and high STAT3 were compared, and differentially expressed transcripts were identified (FDR
    Figure Legend Snippet: Important functional pathways are dysregulated in MDS CD34 + samples with high expression of STAT3. ( A ) Gene expression profiles from samples with low and high STAT3 were compared, and differentially expressed transcripts were identified (FDR

    Techniques Used: Functional Assay, Expressing

    STAT3 is overexpressed in MDS and AML HSCs and progenitors and is associated with worse prognosis. ( A – D ) Gene expression data from sorted MDS/AML bone marrow samples were compared with data from healthy controls (Ctrl) and revealed significantly increased STAT3 expression in LT-HSCs (Lin – , CD34 + , CD38 – , CD90 – , n = 12 MDS/AML, healthy control [HC] = 4), ST-HSCs (Lin – , CD34 + , CD38 – , CD90), and GMPs (Lin – , CD34 + , CD38 + , CD90 + , CD123 + ) ( P
    Figure Legend Snippet: STAT3 is overexpressed in MDS and AML HSCs and progenitors and is associated with worse prognosis. ( A – D ) Gene expression data from sorted MDS/AML bone marrow samples were compared with data from healthy controls (Ctrl) and revealed significantly increased STAT3 expression in LT-HSCs (Lin – , CD34 + , CD38 – , CD90 – , n = 12 MDS/AML, healthy control [HC] = 4), ST-HSCs (Lin – , CD34 + , CD38 – , CD90), and GMPs (Lin – , CD34 + , CD38 + , CD90 + , CD123 + ) ( P

    Techniques Used: Expressing

    5) Product Images from "Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4"

    Article Title: Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4

    Journal: Blood

    doi: 10.1182/blood-2012-09-455204

    Direct transduction of recombinant degradation-resistant HOXB4 protein maintains G-CSF–mobilized CD34 + cells in a more primitive state than wild-type HOXB4. (A) Comparison of HOXB4 mRNA expression levels in CD34 + UCB cells vs CD34 + G-CSF–mobilized
    Figure Legend Snippet: Direct transduction of recombinant degradation-resistant HOXB4 protein maintains G-CSF–mobilized CD34 + cells in a more primitive state than wild-type HOXB4. (A) Comparison of HOXB4 mRNA expression levels in CD34 + UCB cells vs CD34 + G-CSF–mobilized

    Techniques Used: Transduction, Recombinant, Expressing

    6) Product Images from "Superior Human Leukocyte Reconstitution and Susceptibility to Vaginal HIV Transmission in Humanized NOD-scid IL-2R −/− (NSG) BLT Mice"

    Article Title: Superior Human Leukocyte Reconstitution and Susceptibility to Vaginal HIV Transmission in Humanized NOD-scid IL-2R −/− (NSG) BLT Mice

    Journal: Virology

    doi: 10.1016/j.virol.2011.05.013

    Phenotypic analysis of CD34-enriched fetal liver cells. (A) Cryopreserved CD34-enriched fetal liver cells used for injection into BLT mice were immunophenotyped by flow cytometry. Isolated fetal liver cells were stained to determine the frequency of CD34
    Figure Legend Snippet: Phenotypic analysis of CD34-enriched fetal liver cells. (A) Cryopreserved CD34-enriched fetal liver cells used for injection into BLT mice were immunophenotyped by flow cytometry. Isolated fetal liver cells were stained to determine the frequency of CD34

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Cytometry, Isolation, Staining

    7) Product Images from "Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells "

    Article Title: Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells

    Journal: The Journal of Experimental Medicine

    doi:

    Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).
    Figure Legend Snippet: Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).

    Techniques Used: Chemotaxis Assay, Derivative Assay

    RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.
    Figure Legend Snippet: RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Molecular Weight, Derivative Assay

    8) Product Images from "Identifying Regulatory Pathways of SYK Expression in Human Basophils"

    Article Title: Identifying Regulatory Pathways of SYK Expression in Human Basophils

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2019.10.005

    . Using microarray results for 5 types of cells [peripheral blood basophils (PBB), peripheral blood eosinophils (PBE), peripheral blood plasmacytoid dendritic cells (PDC), CD34-derived basophils (CD34B) developed with the G1 or G3 protocols] transcripts were analyzed for following 4 patterns of expression. The short gene name is shown to the left of the heat-map and columns in the heat-map are colored for the ratio of the presence of the transcripts for the comparison shown at the top of the column. The arrows in the patterns indicate whether the ratio for a particular comparison is greater than 1.0, less than 1.0 or relatively unchanged. The colors represent the log-fold-change of the transcript for the particular comparison, ranging from 0.01 to 100, blue to red with shaded green representing changes near 1.0-fold (where unchanged refers to ratios between 0.75- and 1.33-fold).
    Figure Legend Snippet: . Using microarray results for 5 types of cells [peripheral blood basophils (PBB), peripheral blood eosinophils (PBE), peripheral blood plasmacytoid dendritic cells (PDC), CD34-derived basophils (CD34B) developed with the G1 or G3 protocols] transcripts were analyzed for following 4 patterns of expression. The short gene name is shown to the left of the heat-map and columns in the heat-map are colored for the ratio of the presence of the transcripts for the comparison shown at the top of the column. The arrows in the patterns indicate whether the ratio for a particular comparison is greater than 1.0, less than 1.0 or relatively unchanged. The colors represent the log-fold-change of the transcript for the particular comparison, ranging from 0.01 to 100, blue to red with shaded green representing changes near 1.0-fold (where unchanged refers to ratios between 0.75- and 1.33-fold).

    Techniques Used: Microarray, Derivative Assay, Expressing

    9) Product Images from "P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrowhematopoietic stem cells"

    Article Title: P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrowhematopoietic stem cells

    Journal: Genes & Development

    doi: 10.1101/gad.1071503

    Bone-marrow-conditioned medium activates GPR105 altering GPR105 + cell function. ( A ) GPR105 is expressed on the cell surface of transduced cells. COS-7 cells were transiently transfected with HA-epitope-tagged GPR105 ( top panel) or vector alone ( bottom panel) and stained with anti-HA monoclonal antibody without cell permeabilization. Panels are representative fluorophotomicrographs. ( B ) GPR105-transduced cells undergo calcium flux in response to selected conditioned media. GPR105-transfected COS-7 cells were loaded with Fura-2 and assayed using microscopic fluorimetry. Cells were stimulated with the indicated conditioned medium at points indicated by the arrow. Intracellular calcium concentration was monitored by the fluorescence ratio (F340/F380) plotted on the vertical axis. Similar results were obtained in three additional, independent experiments. ( C ) Primary human fetal bone marrow cells that express GPR105 transmigrate in response to bone-marrow-conditioned medium. Transmigration assays using GPR105-enriched CD34 + CD38 – cells or those cells that do not express GPR105 (CD34 + CD38 + ). Results represent the mean and S.E.M. of one of four independent experiments. Migration is expressed as a chemotactic index calculated from the percentage of cells in the test wells passing through a 5-μm filter over 3 h, divided by the percentage of migration in chemokinesis controls. To test for the specificity of transmigration, cells were pretreated with pertussis toxin (P. Toxin), anti-CXCR4 antibody (antiX4), anti-GPR105 antibody (antiGPR105), or control antibody (IgG). Comparison of chemotaxis with and without pretreatment with anti-GPR105 antibody is indicated with the p value following Student's t -test analysis.
    Figure Legend Snippet: Bone-marrow-conditioned medium activates GPR105 altering GPR105 + cell function. ( A ) GPR105 is expressed on the cell surface of transduced cells. COS-7 cells were transiently transfected with HA-epitope-tagged GPR105 ( top panel) or vector alone ( bottom panel) and stained with anti-HA monoclonal antibody without cell permeabilization. Panels are representative fluorophotomicrographs. ( B ) GPR105-transduced cells undergo calcium flux in response to selected conditioned media. GPR105-transfected COS-7 cells were loaded with Fura-2 and assayed using microscopic fluorimetry. Cells were stimulated with the indicated conditioned medium at points indicated by the arrow. Intracellular calcium concentration was monitored by the fluorescence ratio (F340/F380) plotted on the vertical axis. Similar results were obtained in three additional, independent experiments. ( C ) Primary human fetal bone marrow cells that express GPR105 transmigrate in response to bone-marrow-conditioned medium. Transmigration assays using GPR105-enriched CD34 + CD38 – cells or those cells that do not express GPR105 (CD34 + CD38 + ). Results represent the mean and S.E.M. of one of four independent experiments. Migration is expressed as a chemotactic index calculated from the percentage of cells in the test wells passing through a 5-μm filter over 3 h, divided by the percentage of migration in chemokinesis controls. To test for the specificity of transmigration, cells were pretreated with pertussis toxin (P. Toxin), anti-CXCR4 antibody (antiX4), anti-GPR105 antibody (antiGPR105), or control antibody (IgG). Comparison of chemotaxis with and without pretreatment with anti-GPR105 antibody is indicated with the p value following Student's t -test analysis.

    Techniques Used: Cell Function Assay, Transfection, Plasmid Preparation, Staining, Concentration Assay, Fluorescence, Transmigration Assay, Migration, Chemotaxis Assay

    GPR105 is restricted in tissue expression and within hematopoietic cells is limited to primitive cells. ( A ) A human tissue blot with indicated mRNAs was probed with radiolabeled GPR105 cDNA and hybridization determined by autoradiography. Size markers are indicated in kilobases. ( B ) Adult human cell mRNA from cells bearing the indicated phenotype was assessed by poly(A)-primed RT–PCR and the resulting cDNA was probed with either GPR105 or GAPDH sequences. Cells labeled G0 CD34 + CD38 – ) and represent
    Figure Legend Snippet: GPR105 is restricted in tissue expression and within hematopoietic cells is limited to primitive cells. ( A ) A human tissue blot with indicated mRNAs was probed with radiolabeled GPR105 cDNA and hybridization determined by autoradiography. Size markers are indicated in kilobases. ( B ) Adult human cell mRNA from cells bearing the indicated phenotype was assessed by poly(A)-primed RT–PCR and the resulting cDNA was probed with either GPR105 or GAPDH sequences. Cells labeled G0 CD34 + CD38 – ) and represent

    Techniques Used: Expressing, Hybridization, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Labeling

    Bone-marrow-conditioned medium activates GPR105 altering GPR105 + cell function. ( D ) GPR105-transduced primary cord blood CD34 + cells transmigrate in response to conditioned medium from fetal bone marrow stroma. Data are represented as the mean chemotactic index and S.E.M. from six independent experiments with pretreatment conditions as indicated using GFP + cells in each group. Comparison of chemotaxis with and without pretreatment with anti-GPR105 antibody is indicated with the p value following Student's t -test analysis.
    Figure Legend Snippet: Bone-marrow-conditioned medium activates GPR105 altering GPR105 + cell function. ( D ) GPR105-transduced primary cord blood CD34 + cells transmigrate in response to conditioned medium from fetal bone marrow stroma. Data are represented as the mean chemotactic index and S.E.M. from six independent experiments with pretreatment conditions as indicated using GFP + cells in each group. Comparison of chemotaxis with and without pretreatment with anti-GPR105 antibody is indicated with the p value following Student's t -test analysis.

    Techniques Used: Cell Function Assay, Chemotaxis Assay

    Anti-GPR105 recognizes GPR105 and identifies a subset of CD34 + CD38 – fetal bone marrow cells. ( A ) HA-epitope-tagged GPR105 cDNA was in vitro transcribed and translated. ( Left ) In vitro translated protein was immunoprecipitated with anti-GPR105 antibody. Note that the increase of the size in NC-tagged HA is caused by an additional tagging. ( Right ) Similar results were obtained using anti-HA tag monoclonal antibody for immunoprecipitation. PcDNA without GPR105 was used as a negative control. N-HA, N terminus-tagged; C-HA, C terminus-tagged; NC-HA, N and C terminus-tagged. The position of a molecular weight marker (size in kilodaltons) is indicated to the left . ( B ) Flow cytometry of cells transduced with either MSCV control (blue line) or MSCV-GPR105 (red line) vectors and stained with affinity-purified anti-GPR105. ( C ) CD34 + CD38 – GPR105 + human fetal bone marrow cells express GPR105 mRNA as demonstrated by semiquantitative RT–PCR. Cells isolated by cell sorting were assessed using 3 × 10 3 cells of each type and analyzed for GPR105 or actin mRNA; control refers to a no-cDNA template. ( D ) CD34 + CD38 – fetal bone marrow cells stained with anti-GPR105-FITC or control antiserum from a single experiment ( top panels) or composite data from four independent experiments (table).
    Figure Legend Snippet: Anti-GPR105 recognizes GPR105 and identifies a subset of CD34 + CD38 – fetal bone marrow cells. ( A ) HA-epitope-tagged GPR105 cDNA was in vitro transcribed and translated. ( Left ) In vitro translated protein was immunoprecipitated with anti-GPR105 antibody. Note that the increase of the size in NC-tagged HA is caused by an additional tagging. ( Right ) Similar results were obtained using anti-HA tag monoclonal antibody for immunoprecipitation. PcDNA without GPR105 was used as a negative control. N-HA, N terminus-tagged; C-HA, C terminus-tagged; NC-HA, N and C terminus-tagged. The position of a molecular weight marker (size in kilodaltons) is indicated to the left . ( B ) Flow cytometry of cells transduced with either MSCV control (blue line) or MSCV-GPR105 (red line) vectors and stained with affinity-purified anti-GPR105. ( C ) CD34 + CD38 – GPR105 + human fetal bone marrow cells express GPR105 mRNA as demonstrated by semiquantitative RT–PCR. Cells isolated by cell sorting were assessed using 3 × 10 3 cells of each type and analyzed for GPR105 or actin mRNA; control refers to a no-cDNA template. ( D ) CD34 + CD38 – fetal bone marrow cells stained with anti-GPR105-FITC or control antiserum from a single experiment ( top panels) or composite data from four independent experiments (table).

    Techniques Used: In Vitro, Immunoprecipitation, Negative Control, Molecular Weight, Marker, Flow Cytometry, Cytometry, Transduction, Staining, Affinity Purification, Reverse Transcription Polymerase Chain Reaction, Isolation, FACS

    CD34 + CD38 – cells expressing GPR105 are disproportionately in G0 and are enriched for a stem cell functional phenotype. ( D ) Morphology of cells derived from LTC-IC of GPR105 + CD34 + CD38 – cells is consistent with mature myeloid lineage cells. Cells were isolated by micropippetting, stained with May-Grunwald-Giemsa, and assessed by photo-light microscopy. ( E ) Primary cord blood-derived CD34 + cells generate reduced CFC with GPR105 expression. CD34 + cells transduced with MSCV-GPF or MSCV-GFP-GPR105 vector were sorted for GFP expression and assessed in a standard CFC assay. ( F ) Limit dilution CAFC is increased with GPR105 expression in transduced primary CD34 + cells. CD34 + GFP-positive cells as above were evaluated in either CAFC (shown) or LTC-IC assays (data not shown).
    Figure Legend Snippet: CD34 + CD38 – cells expressing GPR105 are disproportionately in G0 and are enriched for a stem cell functional phenotype. ( D ) Morphology of cells derived from LTC-IC of GPR105 + CD34 + CD38 – cells is consistent with mature myeloid lineage cells. Cells were isolated by micropippetting, stained with May-Grunwald-Giemsa, and assessed by photo-light microscopy. ( E ) Primary cord blood-derived CD34 + cells generate reduced CFC with GPR105 expression. CD34 + cells transduced with MSCV-GPF or MSCV-GFP-GPR105 vector were sorted for GFP expression and assessed in a standard CFC assay. ( F ) Limit dilution CAFC is increased with GPR105 expression in transduced primary CD34 + cells. CD34 + GFP-positive cells as above were evaluated in either CAFC (shown) or LTC-IC assays (data not shown).

    Techniques Used: Expressing, Functional Assay, Derivative Assay, Isolation, Staining, Light Microscopy, Transduction, Plasmid Preparation

    CD34 + CD38 – cells expressing GPR105 are disproportionately in G0 and are enriched for a stem cell functional phenotype. ( I ) Cobblestone area formation on 14F1.1 cells ( left ) and myeloid morphology of cells derived from culture of murine lin – GPR105 + cells under myeloid growth conditions ( right ). ( J ) Rag1 and Rag2 expression of murine lin – GPR105 + cells cultured on 14F1.1 cells and under lymphoid cytokine containing methylcellulose. (Lane 1 ) Colonies with and without RT. (Lane 2 ) 14F1.1 stroma cells with and without RT. (Lane 3 ) No template control.
    Figure Legend Snippet: CD34 + CD38 – cells expressing GPR105 are disproportionately in G0 and are enriched for a stem cell functional phenotype. ( I ) Cobblestone area formation on 14F1.1 cells ( left ) and myeloid morphology of cells derived from culture of murine lin – GPR105 + cells under myeloid growth conditions ( right ). ( J ) Rag1 and Rag2 expression of murine lin – GPR105 + cells cultured on 14F1.1 cells and under lymphoid cytokine containing methylcellulose. (Lane 1 ) Colonies with and without RT. (Lane 2 ) 14F1.1 stroma cells with and without RT. (Lane 3 ) No template control.

    Techniques Used: Expressing, Functional Assay, Derivative Assay, Cell Culture

    CD34 + CD38 – cells expressing GPR105 are disproportionately in G0 and are enriched for a stem cell functional phenotype. ( A ) Flow cytometric analysis of PY and Ho staining of human fetal bone marrow cells sorted for CD34 + CD38 – and GPR105; note that events are clustered at the upper limit of the PY axis with the gain set to enhance discrimination of ±PY. The percentage of G0 and G1 cells is indicated, respectively. These data are a representative of two independent experiments with comparable results. ( B ) CFC of GPR105 + versus GPR105 – CD34 + CD38 – cells ( n = 6; p = 0.00003). ( C ) CAFC over time of GPR105 + versus GPR105 – CD34 + CD38 – cells calculated as the ratio relative to week 2 ( top panel). The fractional proportion of week 2 colonies is provided in the accompanying table. Cells were plated at 3–6 twofold dilutions in replicate wells and scored each week. Data shown are one representative of three independent experiments with comparable results.
    Figure Legend Snippet: CD34 + CD38 – cells expressing GPR105 are disproportionately in G0 and are enriched for a stem cell functional phenotype. ( A ) Flow cytometric analysis of PY and Ho staining of human fetal bone marrow cells sorted for CD34 + CD38 – and GPR105; note that events are clustered at the upper limit of the PY axis with the gain set to enhance discrimination of ±PY. The percentage of G0 and G1 cells is indicated, respectively. These data are a representative of two independent experiments with comparable results. ( B ) CFC of GPR105 + versus GPR105 – CD34 + CD38 – cells ( n = 6; p = 0.00003). ( C ) CAFC over time of GPR105 + versus GPR105 – CD34 + CD38 – cells calculated as the ratio relative to week 2 ( top panel). The fractional proportion of week 2 colonies is provided in the accompanying table. Cells were plated at 3–6 twofold dilutions in replicate wells and scored each week. Data shown are one representative of three independent experiments with comparable results.

    Techniques Used: Expressing, Functional Assay, Flow Cytometry, Staining

    CD34 + CD38 – cells expressing GPR105 are disproportionately in G0 and are enriched for a stem cell functional phenotype. ( G ) Cell cycle analysis of transduced cord blood CD34 + cells. GFP + cells transduced as above were assessed for cell cycle status by Hoechst 33342 staining. ( H ) Cells from murine fetal bone marrow and liver were stained for surface markers of lineage, Sca-1, c-Kit receptor, and GPR105. Lin – Sca-1 + cells were then gated, and the expression of c-Kit and GPR105 was analyzed. The percentages in the upper left quadrant represent the proportion of the lin – Sca-1 + cells that are GPR105 + . Data represent one of three experiments with comparable results.
    Figure Legend Snippet: CD34 + CD38 – cells expressing GPR105 are disproportionately in G0 and are enriched for a stem cell functional phenotype. ( G ) Cell cycle analysis of transduced cord blood CD34 + cells. GFP + cells transduced as above were assessed for cell cycle status by Hoechst 33342 staining. ( H ) Cells from murine fetal bone marrow and liver were stained for surface markers of lineage, Sca-1, c-Kit receptor, and GPR105. Lin – Sca-1 + cells were then gated, and the expression of c-Kit and GPR105 was analyzed. The percentages in the upper left quadrant represent the proportion of the lin – Sca-1 + cells that are GPR105 + . Data represent one of three experiments with comparable results.

    Techniques Used: Expressing, Functional Assay, Cell Cycle Assay, Staining

    10) Product Images from "Dynamic Expression of Specific miRNAs during Erythroid Differentiation of Human Embryonic Stem Cells"

    Article Title: Dynamic Expression of Specific miRNAs during Erythroid Differentiation of Human Embryonic Stem Cells

    Journal: Molecules and Cells

    doi: 10.1007/s10059-012-0090-6

    Flow cytometric analysis of erythroid cells developed from hESC-derived CD34+ cells. Expression of CD71 and GPA-1, erythrocyte markers, on erythroid cells developed from hESC-derived CD34+ cells at two different phases (day 7 and day 14) was determined.
    Figure Legend Snippet: Flow cytometric analysis of erythroid cells developed from hESC-derived CD34+ cells. Expression of CD71 and GPA-1, erythrocyte markers, on erythroid cells developed from hESC-derived CD34+ cells at two different phases (day 7 and day 14) was determined.

    Techniques Used: Flow Cytometry, Derivative Assay, Expressing

    Differentiation of hESC derived CD34+ cells into erythroid cells. hESC, hESC-derived CD34+ cells and CD34+ cells-derived erythrocyte progenitors at two stages were produced as described in and CD34 and GPA-1 mRNA determined by RT-PCR. (A). The
    Figure Legend Snippet: Differentiation of hESC derived CD34+ cells into erythroid cells. hESC, hESC-derived CD34+ cells and CD34+ cells-derived erythrocyte progenitors at two stages were produced as described in and CD34 and GPA-1 mRNA determined by RT-PCR. (A). The

    Techniques Used: Derivative Assay, Produced, Reverse Transcription Polymerase Chain Reaction

    Culture conditions under which H9 hESC differentiated into CD34+ hematopoietic cells and subsequently into erythroid cells. OP9 cells and hBM derived MSC cells were used as feeder cells for differentiation of H9 hESCs into CD34+ cells and subsequent erythroid
    Figure Legend Snippet: Culture conditions under which H9 hESC differentiated into CD34+ hematopoietic cells and subsequently into erythroid cells. OP9 cells and hBM derived MSC cells were used as feeder cells for differentiation of H9 hESCs into CD34+ cells and subsequent erythroid

    Techniques Used: Derivative Assay

    Development of CD34+ cells expressing hematopoietic markers from hESCs. Initial hESCs and cells differentiated from hESC were compared for the mRNA expression levels of OCT4, CD34, GATA4, AFP, DCN, Brachury and NeuroD1 mRNA after a 10-day co-culture with
    Figure Legend Snippet: Development of CD34+ cells expressing hematopoietic markers from hESCs. Initial hESCs and cells differentiated from hESC were compared for the mRNA expression levels of OCT4, CD34, GATA4, AFP, DCN, Brachury and NeuroD1 mRNA after a 10-day co-culture with

    Techniques Used: Expressing, Co-Culture Assay

    Differential miRNAs expression in CB derived CD34+ cells. CD34+ cells, hESC, hEB, MSC and WI-38 cells were subject to Genopal miRNA microarray assays and predominant species of miRNAs were identified. Among various miRNAs miR-142-3p, miR-142-5p, miR-146a
    Figure Legend Snippet: Differential miRNAs expression in CB derived CD34+ cells. CD34+ cells, hESC, hEB, MSC and WI-38 cells were subject to Genopal miRNA microarray assays and predominant species of miRNAs were identified. Among various miRNAs miR-142-3p, miR-142-5p, miR-146a

    Techniques Used: Expressing, Derivative Assay, Microarray

    Induction of four specific miRNAs expression in generated erythroid cells. hESC-derived CD34+ cells were induced to differentiate into erythroid cells in the presence of SCF, IL-3 and EPO (phase I), and EPO with MSC, feeder cells (phase II). Expression
    Figure Legend Snippet: Induction of four specific miRNAs expression in generated erythroid cells. hESC-derived CD34+ cells were induced to differentiate into erythroid cells in the presence of SCF, IL-3 and EPO (phase I), and EPO with MSC, feeder cells (phase II). Expression

    Techniques Used: Expressing, Generated, Derivative Assay

    11) Product Images from "Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells "

    Article Title: Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells

    Journal: The Journal of Experimental Medicine

    doi:

    Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).
    Figure Legend Snippet: Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).

    Techniques Used: Chemotaxis Assay, Derivative Assay

    RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.
    Figure Legend Snippet: RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Molecular Weight, Derivative Assay

    12) Product Images from "Identifying Regulatory Pathways of SYK Expression in Human Basophils"

    Article Title: Identifying Regulatory Pathways of SYK Expression in Human Basophils

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2019.10.005

    . Using microarray results for 5 types of cells [peripheral blood basophils (PBB), peripheral blood eosinophils (PBE), peripheral blood plasmacytoid dendritic cells (PDC), CD34-derived basophils (CD34B) developed with the G1 or G3 protocols] transcripts were analyzed for following 4 patterns of expression. The short gene name is shown to the left of the heat-map and columns in the heat-map are colored for the ratio of the presence of the transcripts for the comparison shown at the top of the column. The arrows in the patterns indicate whether the ratio for a particular comparison is greater than 1.0, less than 1.0 or relatively unchanged. The colors represent the log-fold-change of the transcript for the particular comparison, ranging from 0.01 to 100, blue to red with shaded green representing changes near 1.0-fold (where unchanged refers to ratios between 0.75- and 1.33-fold).
    Figure Legend Snippet: . Using microarray results for 5 types of cells [peripheral blood basophils (PBB), peripheral blood eosinophils (PBE), peripheral blood plasmacytoid dendritic cells (PDC), CD34-derived basophils (CD34B) developed with the G1 or G3 protocols] transcripts were analyzed for following 4 patterns of expression. The short gene name is shown to the left of the heat-map and columns in the heat-map are colored for the ratio of the presence of the transcripts for the comparison shown at the top of the column. The arrows in the patterns indicate whether the ratio for a particular comparison is greater than 1.0, less than 1.0 or relatively unchanged. The colors represent the log-fold-change of the transcript for the particular comparison, ranging from 0.01 to 100, blue to red with shaded green representing changes near 1.0-fold (where unchanged refers to ratios between 0.75- and 1.33-fold).

    Techniques Used: Microarray, Derivative Assay, Expressing

    13) Product Images from "Neonatal mucosal immunization with a non-living, non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection"

    Article Title: Neonatal mucosal immunization with a non-living, non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection

    Journal: Mucosal immunology

    doi: 10.1038/mi.2009.131

    GEM particles enhanced the maturation of neonatal and adult human DC and the stimulation of T cells in a MLR. ( a ) Expression of cell surface markers CD80, CD86, CD83 and HLA-DR on human CD11c + neonatal and adult DC stimulated with GEM particles (shaded area) or mock- treated (solid line); the dashed line indicates the isotype control. The MFI on CD11c + gated cells is indicated. ( b ) Individual (i–v) and Z-stack projection (vi) confocal laser microscopy images showing CD11c + DC from human newborns harboring FITC-labeled GEM particles (arrows). CD11c + cell-surface expression is shown in red, and nuclei are shown by blue fluorescent staining. Scale bars, 2 μm. ( c ) Cytokines produced by human neonatal CD34 + - and adult derived DC stimulated with GEM particles or mock-stimulated and measured in culture supernatants. Data represent mean cytokine concentration±s.e.m. from two independent experiments. ( d ) FITC-dextran uptake by neonatal CD11c + and adult human DC exposed to GEM or TNF-α (positive control), or mock-treated DC, measured by flow cytometry; data represents MFI±s.e.m. from three independent experiments. ( e ) Allogeneic stimulation of adult CD3 + T cells in the presence of neonatal and adult human DC stimulated with GEM particles or TNF-α, or mock-stimulated DC. Data show mean cpm±s.e.m. from one of two independent experiments. ( c–e ) Significant differences (* P
    Figure Legend Snippet: GEM particles enhanced the maturation of neonatal and adult human DC and the stimulation of T cells in a MLR. ( a ) Expression of cell surface markers CD80, CD86, CD83 and HLA-DR on human CD11c + neonatal and adult DC stimulated with GEM particles (shaded area) or mock- treated (solid line); the dashed line indicates the isotype control. The MFI on CD11c + gated cells is indicated. ( b ) Individual (i–v) and Z-stack projection (vi) confocal laser microscopy images showing CD11c + DC from human newborns harboring FITC-labeled GEM particles (arrows). CD11c + cell-surface expression is shown in red, and nuclei are shown by blue fluorescent staining. Scale bars, 2 μm. ( c ) Cytokines produced by human neonatal CD34 + - and adult derived DC stimulated with GEM particles or mock-stimulated and measured in culture supernatants. Data represent mean cytokine concentration±s.e.m. from two independent experiments. ( d ) FITC-dextran uptake by neonatal CD11c + and adult human DC exposed to GEM or TNF-α (positive control), or mock-treated DC, measured by flow cytometry; data represents MFI±s.e.m. from three independent experiments. ( e ) Allogeneic stimulation of adult CD3 + T cells in the presence of neonatal and adult human DC stimulated with GEM particles or TNF-α, or mock-stimulated DC. Data show mean cpm±s.e.m. from one of two independent experiments. ( c–e ) Significant differences (* P

    Techniques Used: Expressing, Microscopy, Labeling, Staining, Produced, Derivative Assay, Concentration Assay, Positive Control, Flow Cytometry, Cytometry

    14) Product Images from "Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells "

    Article Title: Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells

    Journal: The Journal of Experimental Medicine

    doi:

    Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).
    Figure Legend Snippet: Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).

    Techniques Used: Chemotaxis Assay, Derivative Assay

    RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.
    Figure Legend Snippet: RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Molecular Weight, Derivative Assay

    15) Product Images from "Polyurethane scaffolds seeded with CD34+ cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors"

    Article Title: Polyurethane scaffolds seeded with CD34+ cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors

    Journal: Scientific Reports

    doi: 10.1038/srep32149

    Cell surface expression during standard erythroid and megakaryocyte 2D cultures. ( A,B ) Immunophenotype of cells analysed daily from standard 2D erythroid and megakaryocyte cultures expressed as a percentage of the live cell population for each of the following cell surface markers; GPA + (APC), CD61 + (APC Vio770) and CD34 + (VioBlue). Erythroid cultures are first cultured in expansion medium and then differentiation medium from day 7, whereas megakaryocyte cultures remain in the same megakaryocyte culture medium throughout. ( A ) Erythroid culture, ( B ) megakaryocyte culture. ( C ) Expression of mature surface markers band 3 (Bric71) and CD41 (5B12) in both erythroid (blasts) and megakaryocyte (megs) cultures on days 3, 8 and 11 of culture; monoclonal primary antibodies were coupled to an APC secondary antibody. Expression is shown as a percentage of the total live cell population. N = 3 independent experiments with error bars representing the standard error of the mean. ( D ) Daily analysis of megakaryocyte and erythroid cultures as assessed with flow cytometry for 11 days with the following antibodies; CD34, CD36, CD61, CD14 and GPA. Above erythroid, below megakaryocyte culture. Schematic is a representation of the duration of expression in addition to the level of expression for each cell surface marker. N = 3 independent experiments.
    Figure Legend Snippet: Cell surface expression during standard erythroid and megakaryocyte 2D cultures. ( A,B ) Immunophenotype of cells analysed daily from standard 2D erythroid and megakaryocyte cultures expressed as a percentage of the live cell population for each of the following cell surface markers; GPA + (APC), CD61 + (APC Vio770) and CD34 + (VioBlue). Erythroid cultures are first cultured in expansion medium and then differentiation medium from day 7, whereas megakaryocyte cultures remain in the same megakaryocyte culture medium throughout. ( A ) Erythroid culture, ( B ) megakaryocyte culture. ( C ) Expression of mature surface markers band 3 (Bric71) and CD41 (5B12) in both erythroid (blasts) and megakaryocyte (megs) cultures on days 3, 8 and 11 of culture; monoclonal primary antibodies were coupled to an APC secondary antibody. Expression is shown as a percentage of the total live cell population. N = 3 independent experiments with error bars representing the standard error of the mean. ( D ) Daily analysis of megakaryocyte and erythroid cultures as assessed with flow cytometry for 11 days with the following antibodies; CD34, CD36, CD61, CD14 and GPA. Above erythroid, below megakaryocyte culture. Schematic is a representation of the duration of expression in addition to the level of expression for each cell surface marker. N = 3 independent experiments.

    Techniques Used: Expressing, Cell Culture, Flow Cytometry, Cytometry, Marker

    Parallel control cultures grown in the absence of scaffolds are sustained for the duration of the culture period however cell death is increased in controls compared to scaffold cultures. ( A ) Cell counts of high-density control cultures seeded with 0.5 × 10 6 CD34 + cells and maintained in 1.5 mL either BSFEM or with the addition of EPO or TPO. Dead cells were excluded using propidium iodide. ( B ) The average cumulative egress for scaffolds and the maximal point of control cultures, taken as day 12 for BSFEM, +EPO and +TPO. Each culture was seeded with 0.5 × 10 6 CD34 + cells on day 0. The difference between the cumulative scaffold egress and peak of control cultures grown in the absence of scaffolds was found to be statistically significant in the plus TPO condition (P = 0.00557) but not in plus EPO (P = 0.05564) or BSFEM (P = 0.06647). ( C ) Cell death was assessed using propidium iodide as per manufacturers instructions and expressed as percentage cell death across the BSFEM, +EPO and +TPO conditions in both scaffold egress (red) and control cultures grown in the absence of scaffolds (blue), every second day for the 28 day period. Cell death was found to be statistically significant between control and scaffold cultures in BSFEM and upon the addition of TPO (P = ≤ 0.05). N = 3 independent experiments in at least duplicate for PU scaffolds, error bars represent the standard error of the mean and significance was tested using the students T-test.
    Figure Legend Snippet: Parallel control cultures grown in the absence of scaffolds are sustained for the duration of the culture period however cell death is increased in controls compared to scaffold cultures. ( A ) Cell counts of high-density control cultures seeded with 0.5 × 10 6 CD34 + cells and maintained in 1.5 mL either BSFEM or with the addition of EPO or TPO. Dead cells were excluded using propidium iodide. ( B ) The average cumulative egress for scaffolds and the maximal point of control cultures, taken as day 12 for BSFEM, +EPO and +TPO. Each culture was seeded with 0.5 × 10 6 CD34 + cells on day 0. The difference between the cumulative scaffold egress and peak of control cultures grown in the absence of scaffolds was found to be statistically significant in the plus TPO condition (P = 0.00557) but not in plus EPO (P = 0.05564) or BSFEM (P = 0.06647). ( C ) Cell death was assessed using propidium iodide as per manufacturers instructions and expressed as percentage cell death across the BSFEM, +EPO and +TPO conditions in both scaffold egress (red) and control cultures grown in the absence of scaffolds (blue), every second day for the 28 day period. Cell death was found to be statistically significant between control and scaffold cultures in BSFEM and upon the addition of TPO (P = ≤ 0.05). N = 3 independent experiments in at least duplicate for PU scaffolds, error bars represent the standard error of the mean and significance was tested using the students T-test.

    Techniques Used:

    CD34 + cells persist to the culture endpoint within the honeycomb structure of PU scaffolds. Polyurethane scaffolds were cultured for 28 days and paraffin wax blocks cut to 10 μm sections. Scaffold sections were probed with CD44 (Bric 235), CD34 (Birma K3), GPA (Bric 256), CD42b (BioLegend), and a macrophage marker (calprotectin) and coupled to Alexa 647 secondary antibody (white), DAPI was used to stain the nucleus (blue). Images were acquired using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope using the 60x lens (N.A. 1.4), scale bars are 27 μm, images are representative and N = 3.
    Figure Legend Snippet: CD34 + cells persist to the culture endpoint within the honeycomb structure of PU scaffolds. Polyurethane scaffolds were cultured for 28 days and paraffin wax blocks cut to 10 μm sections. Scaffold sections were probed with CD44 (Bric 235), CD34 (Birma K3), GPA (Bric 256), CD42b (BioLegend), and a macrophage marker (calprotectin) and coupled to Alexa 647 secondary antibody (white), DAPI was used to stain the nucleus (blue). Images were acquired using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope using the 60x lens (N.A. 1.4), scale bars are 27 μm, images are representative and N = 3.

    Techniques Used: Cell Culture, Marker, Staining, Laser-Scanning Microscopy, Inverted Epifluorescence

    Flow cytometry analysis reveals the cells that egress from the scaffolds are largely early erythroid progenitors. Cell populations that egress from scaffold cultures were assessed every 4 days using antibodies to detect the following populations; CD14 (FITC), CD34 (VioBlue), CD36 (PE), CD61 (APC-Vio770) and GPA (APC). Cellular populations are shown as a percentage of the total live cell population for each condition, plus EPO, plus TPO and BSFEM. N = 3 independent experiments in at least duplicate, error bars represent the standard error of the mean.
    Figure Legend Snippet: Flow cytometry analysis reveals the cells that egress from the scaffolds are largely early erythroid progenitors. Cell populations that egress from scaffold cultures were assessed every 4 days using antibodies to detect the following populations; CD14 (FITC), CD34 (VioBlue), CD36 (PE), CD61 (APC-Vio770) and GPA (APC). Cellular populations are shown as a percentage of the total live cell population for each condition, plus EPO, plus TPO and BSFEM. N = 3 independent experiments in at least duplicate, error bars represent the standard error of the mean.

    Techniques Used: Flow Cytometry, Cytometry

    Polyurethane and bone donor scaffolds sustain cellular egress for a 28-day period. ( A ) Scanning electron micrographs (SEM) of the human bone donor (left) and polyurethane (PU) (right) scaffolds, scale bars are 1 mm and 500 μm respectively. ( B ) Schematic of the scaffold culture protocol. Scaffolds are seeded with 0.5 × 10 6 lineage depleted or CD34 + cells on day 0 in 100 μL of BSFEM for 2 hours before being immersed in 1.5 mL of medium. Scaffolds are transferred to a fresh well of medium every 2 days and the output is formed from the previous well. The process continues for the 28 day culture period. ( C ) Cell counts of egress from PU scaffolds seeded with CD34 + cells (blue), bone donor scaffolds seeded with CD34 + cells (red) and PU scaffolds seeded with lineage depleted cells (green) cultured in BSFEM with the addition of EPO, across the 28 day culture period. ( D ) Cumulative egress from each scaffold in ( C ) over the entire 28 day culture period ( E ) Cell counts of egress from PU scaffolds seeded with CD34 + cells in BSFEM (red) or with EPO (blue) or TPO (green) across the 28 day culture period. ( F ) Cumulative egress from each scaffolds in ( E ) over the entire 28 day culture period. Outputs for the CD34 + plus EPO experiments in graphs ( C–F) are derived from a single set of experiments. N = 3 independent experiments in at least duplicate for PU scaffolds, N = 2 in triplicate for human bone donor scaffolds. Error bars represent the standard error of the mean. Significance was tested using students T-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
    Figure Legend Snippet: Polyurethane and bone donor scaffolds sustain cellular egress for a 28-day period. ( A ) Scanning electron micrographs (SEM) of the human bone donor (left) and polyurethane (PU) (right) scaffolds, scale bars are 1 mm and 500 μm respectively. ( B ) Schematic of the scaffold culture protocol. Scaffolds are seeded with 0.5 × 10 6 lineage depleted or CD34 + cells on day 0 in 100 μL of BSFEM for 2 hours before being immersed in 1.5 mL of medium. Scaffolds are transferred to a fresh well of medium every 2 days and the output is formed from the previous well. The process continues for the 28 day culture period. ( C ) Cell counts of egress from PU scaffolds seeded with CD34 + cells (blue), bone donor scaffolds seeded with CD34 + cells (red) and PU scaffolds seeded with lineage depleted cells (green) cultured in BSFEM with the addition of EPO, across the 28 day culture period. ( D ) Cumulative egress from each scaffold in ( C ) over the entire 28 day culture period ( E ) Cell counts of egress from PU scaffolds seeded with CD34 + cells in BSFEM (red) or with EPO (blue) or TPO (green) across the 28 day culture period. ( F ) Cumulative egress from each scaffolds in ( E ) over the entire 28 day culture period. Outputs for the CD34 + plus EPO experiments in graphs ( C–F) are derived from a single set of experiments. N = 3 independent experiments in at least duplicate for PU scaffolds, N = 2 in triplicate for human bone donor scaffolds. Error bars represent the standard error of the mean. Significance was tested using students T-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

    Techniques Used: Cell Culture, Derivative Assay

    16) Product Images from "Interplay of macrophages and T cells in the lung vasculature"

    Article Title: Interplay of macrophages and T cells in the lung vasculature

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00357.2011

    Immunofluorescent detection of cell surface markers, CD117 (c-kit), factor VIII (F VIII), VEGFR2, Lycopersicon esculentum lectin, CD31 (platelet endothelial cell adhesion molecule-1), and cell-cell adhesion glycoprotein CD34 in HUVEC and RAW 264.7 cells.
    Figure Legend Snippet: Immunofluorescent detection of cell surface markers, CD117 (c-kit), factor VIII (F VIII), VEGFR2, Lycopersicon esculentum lectin, CD31 (platelet endothelial cell adhesion molecule-1), and cell-cell adhesion glycoprotein CD34 in HUVEC and RAW 264.7 cells.

    Techniques Used:

    17) Product Images from "Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model"

    Article Title: Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2014.51

    Vector integration alters cellular gene expression. Expression of the gene close to the insertion site was determined by qPCR: ( a ) ANGPT1 in BM samples of mouse M69 (left, arrow); ANGPT2 in BM samples of mouse M53 (middle); PMVK in BM samples of mouse M65 (right). Expression levels were normalized to mobilized peripheral blood CD34 + cells, CD8 + T-cells served as a negative control (mean ± SD). The expression was further compared to other mice of the study with different integration sites (mean ± SD). Student's t -test: ** P
    Figure Legend Snippet: Vector integration alters cellular gene expression. Expression of the gene close to the insertion site was determined by qPCR: ( a ) ANGPT1 in BM samples of mouse M69 (left, arrow); ANGPT2 in BM samples of mouse M53 (middle); PMVK in BM samples of mouse M65 (right). Expression levels were normalized to mobilized peripheral blood CD34 + cells, CD8 + T-cells served as a negative control (mean ± SD). The expression was further compared to other mice of the study with different integration sites (mean ± SD). Student's t -test: ** P

    Techniques Used: Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Negative Control, Mouse Assay

    In vitro characteristics of expanded CD34 + cells. ( a ) Cord blood-derived CD34 + cells were expanded with four different cytokine conditions for 10 days, the total cell numbers counted and the fold expansion of total cells calculated (mean ± SD, n = 4). ( b ) Expansion of CD34 marker positive cells after 10 days of culture in the four different cytokine conditions (mean ± SD, n = 3). ( c ) Representative flow cytometric analysis of CD34 marker expression after four (STF d4) and 10 days of culture in the different cytokine conditions compared to the expression on CD34+ enriched and uncultured cord blood cells. ( d ) Contribution (%) of CD34 + cells in four and 10 days expanded cells in comparison to day 0 cells (mean ± SD, n = 3). ( e ) CD34 expression levels, as measured by the Mean fluorescence intensity (MFI), on cultured cells at days 4 and 10 correlated to the expression level (MFI) in cells cultured with STF at the respective day of analysis (this value set to one, mean ± SD, n = 3). ( f ) Number of colonies of transduced and expanded CB-CD34 + cells plated after 4 and 10 days of culture (mean ± SD), compared to cells plated without in vitro culture (black column). Colony numbers per 1 × 10 3 cells plated cells/assay, three experiments were analyzed each, in triplicate assays. ( g ) Fold increase of colony forming cells in the cultures during the 4 and 10 days of culture. * P
    Figure Legend Snippet: In vitro characteristics of expanded CD34 + cells. ( a ) Cord blood-derived CD34 + cells were expanded with four different cytokine conditions for 10 days, the total cell numbers counted and the fold expansion of total cells calculated (mean ± SD, n = 4). ( b ) Expansion of CD34 marker positive cells after 10 days of culture in the four different cytokine conditions (mean ± SD, n = 3). ( c ) Representative flow cytometric analysis of CD34 marker expression after four (STF d4) and 10 days of culture in the different cytokine conditions compared to the expression on CD34+ enriched and uncultured cord blood cells. ( d ) Contribution (%) of CD34 + cells in four and 10 days expanded cells in comparison to day 0 cells (mean ± SD, n = 3). ( e ) CD34 expression levels, as measured by the Mean fluorescence intensity (MFI), on cultured cells at days 4 and 10 correlated to the expression level (MFI) in cells cultured with STF at the respective day of analysis (this value set to one, mean ± SD, n = 3). ( f ) Number of colonies of transduced and expanded CB-CD34 + cells plated after 4 and 10 days of culture (mean ± SD), compared to cells plated without in vitro culture (black column). Colony numbers per 1 × 10 3 cells plated cells/assay, three experiments were analyzed each, in triplicate assays. ( g ) Fold increase of colony forming cells in the cultures during the 4 and 10 days of culture. * P

    Techniques Used: In Vitro, Derivative Assay, Marker, Flow Cytometry, Expressing, Fluorescence, Cell Culture

    18) Product Images from "GM-CSF Promotes the Expansion and Differentiation of Cord Blood Myeloid-Derived Suppressor Cells, Which Attenuate Xenogeneic Graft-vs.-Host Disease"

    Article Title: GM-CSF Promotes the Expansion and Differentiation of Cord Blood Myeloid-Derived Suppressor Cells, Which Attenuate Xenogeneic Graft-vs.-Host Disease

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00183

    Expansion of CB CD34 + cells according to cytokine combinations and differentiation into MDSCs. (A) The scheme of cell expansion or differentiation from CB CD34 + cells. (B) Fold expansion by different cytokine combinations. The culture medium was replaced with each cytokine combinations every week. (C) The FITC conjugated anti-human CD33 were compensated with compensation bead to avoid spillover with PE conjugated anti-human CD11b. (D) Expression of CD11b + CD33 + . The cells were stained with fluorochrome conjugated anti-human CD33 (FITC) and aniti-human CD11b (PE) every week. (E) The CD34 + cells were cultured with human GM-CSF (100 ng/ml) and human SCF (50 ng/ml) for 3 weeks and the cells were stained like (D) . The stained cells were sorted by FACS Aria and the sorted cells (CD11b + CD33b + vs. CD11b − CD33 + ) were cultured with human GM-CSF (100ng/ml) and human SCF (50ng/ml) for a further 1 week. After one week, the sorted cells were stained like (D) and analyzed by flow cytometry. (F) Expression of HLA-DR, CD14 and CD15. The cells were stained with efluor450-conjugated anti-human HLA-DR, PE-Cy7 anti-human CD14 and APC anti-human CD15 from 3 to 6 weeks, respectively and analyzed by flow cytometry. These experiments were reproduced in 10 individuals (from B to D and F ) and 6 individuals (E) .
    Figure Legend Snippet: Expansion of CB CD34 + cells according to cytokine combinations and differentiation into MDSCs. (A) The scheme of cell expansion or differentiation from CB CD34 + cells. (B) Fold expansion by different cytokine combinations. The culture medium was replaced with each cytokine combinations every week. (C) The FITC conjugated anti-human CD33 were compensated with compensation bead to avoid spillover with PE conjugated anti-human CD11b. (D) Expression of CD11b + CD33 + . The cells were stained with fluorochrome conjugated anti-human CD33 (FITC) and aniti-human CD11b (PE) every week. (E) The CD34 + cells were cultured with human GM-CSF (100 ng/ml) and human SCF (50 ng/ml) for 3 weeks and the cells were stained like (D) . The stained cells were sorted by FACS Aria and the sorted cells (CD11b + CD33b + vs. CD11b − CD33 + ) were cultured with human GM-CSF (100ng/ml) and human SCF (50ng/ml) for a further 1 week. After one week, the sorted cells were stained like (D) and analyzed by flow cytometry. (F) Expression of HLA-DR, CD14 and CD15. The cells were stained with efluor450-conjugated anti-human HLA-DR, PE-Cy7 anti-human CD14 and APC anti-human CD15 from 3 to 6 weeks, respectively and analyzed by flow cytometry. These experiments were reproduced in 10 individuals (from B to D and F ) and 6 individuals (E) .

    Techniques Used: Expressing, Staining, Cell Culture, FACS, Flow Cytometry, Cytometry

    19) Product Images from "Identifying Regulatory Pathways of SYK Expression in Human Basophils"

    Article Title: Identifying Regulatory Pathways of SYK Expression in Human Basophils

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2019.10.005

    . Using microarray results for 5 types of cells [peripheral blood basophils (PBB), peripheral blood eosinophils (PBE), peripheral blood plasmacytoid dendritic cells (PDC), CD34-derived basophils (CD34B) developed with the G1 or G3 protocols] transcripts were analyzed for following 4 patterns of expression. The short gene name is shown to the left of the heat-map and columns in the heat-map are colored for the ratio of the presence of the transcripts for the comparison shown at the top of the column. The arrows in the patterns indicate whether the ratio for a particular comparison is greater than 1.0, less than 1.0 or relatively unchanged. The colors represent the log-fold-change of the transcript for the particular comparison, ranging from 0.01 to 100, blue to red with shaded green representing changes near 1.0-fold (where unchanged refers to ratios between 0.75- and 1.33-fold).
    Figure Legend Snippet: . Using microarray results for 5 types of cells [peripheral blood basophils (PBB), peripheral blood eosinophils (PBE), peripheral blood plasmacytoid dendritic cells (PDC), CD34-derived basophils (CD34B) developed with the G1 or G3 protocols] transcripts were analyzed for following 4 patterns of expression. The short gene name is shown to the left of the heat-map and columns in the heat-map are colored for the ratio of the presence of the transcripts for the comparison shown at the top of the column. The arrows in the patterns indicate whether the ratio for a particular comparison is greater than 1.0, less than 1.0 or relatively unchanged. The colors represent the log-fold-change of the transcript for the particular comparison, ranging from 0.01 to 100, blue to red with shaded green representing changes near 1.0-fold (where unchanged refers to ratios between 0.75- and 1.33-fold).

    Techniques Used: Microarray, Derivative Assay, Expressing

    20) Product Images from "Identifying Regulatory Pathways of SYK Expression in Human Basophils"

    Article Title: Identifying Regulatory Pathways of SYK Expression in Human Basophils

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2019.10.005

    . Using microarray results for 5 types of cells [peripheral blood basophils (PBB), peripheral blood eosinophils (PBE), peripheral blood plasmacytoid dendritic cells (PDC), CD34-derived basophils (CD34B) developed with the G1 or G3 protocols] transcripts were analyzed for following 4 patterns of expression. The short gene name is shown to the left of the heat-map and columns in the heat-map are colored for the ratio of the presence of the transcripts for the comparison shown at the top of the column. The arrows in the patterns indicate whether the ratio for a particular comparison is greater than 1.0, less than 1.0 or relatively unchanged. The colors represent the log-fold-change of the transcript for the particular comparison, ranging from 0.01 to 100, blue to red with shaded green representing changes near 1.0-fold (where unchanged refers to ratios between 0.75- and 1.33-fold).
    Figure Legend Snippet: . Using microarray results for 5 types of cells [peripheral blood basophils (PBB), peripheral blood eosinophils (PBE), peripheral blood plasmacytoid dendritic cells (PDC), CD34-derived basophils (CD34B) developed with the G1 or G3 protocols] transcripts were analyzed for following 4 patterns of expression. The short gene name is shown to the left of the heat-map and columns in the heat-map are colored for the ratio of the presence of the transcripts for the comparison shown at the top of the column. The arrows in the patterns indicate whether the ratio for a particular comparison is greater than 1.0, less than 1.0 or relatively unchanged. The colors represent the log-fold-change of the transcript for the particular comparison, ranging from 0.01 to 100, blue to red with shaded green representing changes near 1.0-fold (where unchanged refers to ratios between 0.75- and 1.33-fold).

    Techniques Used: Microarray, Derivative Assay, Expressing

    21) Product Images from "Stem and progenitor cells in myelodysplastic syndromes show aberrant stage-specific expansion and harbor genetic and epigenetic alterations"

    Article Title: Stem and progenitor cells in myelodysplastic syndromes show aberrant stage-specific expansion and harbor genetic and epigenetic alterations

    Journal: Blood

    doi: 10.1182/blood-2011-12-399683

    Cytogenetic alterations are seen in MDS HSCs. (A) Colony formation assay using sorted Lin − CD34 + CD38 − cells from 4 MDS patients and 2 healthy controls. Data are mean ± SD for colonies arising from BFU-E and CFU-GM per 5000 plated
    Figure Legend Snippet: Cytogenetic alterations are seen in MDS HSCs. (A) Colony formation assay using sorted Lin − CD34 + CD38 − cells from 4 MDS patients and 2 healthy controls. Data are mean ± SD for colonies arising from BFU-E and CFU-GM per 5000 plated

    Techniques Used: Colony Assay

    MDS BM shows expanded stem and progenitor populations. (A) Representative samples of lower- and higher-risk MDS and a healthy control. Shown are FACS analyses of anti-CD34/CD38 costainings within CD34-enriched, viable, lineage marker-negative BM mononuclear
    Figure Legend Snippet: MDS BM shows expanded stem and progenitor populations. (A) Representative samples of lower- and higher-risk MDS and a healthy control. Shown are FACS analyses of anti-CD34/CD38 costainings within CD34-enriched, viable, lineage marker-negative BM mononuclear

    Techniques Used: FACS, Marker

    22) Product Images from "GATA2−/− human ESCs undergo attenuated endothelial to hematopoietic transition and thereafter granulocyte commitment"

    Article Title: GATA2−/− human ESCs undergo attenuated endothelial to hematopoietic transition and thereafter granulocyte commitment

    Journal: Cell Regeneration

    doi: 10.1186/s13619-015-0018-7

    Forced expression of SPI1 in H1- GATA2 −/− restores the generation of granulocytes upon OP9 co-culture. a , b Diagram of the strategy of SPI1 rescue experiments. SPI1 linked with a puromycin resistance gene by T2A sequence was controlled by a Dox-inducible promoter in lentiviral-based vectors for Dox-inducible expression of SPI1. The expression of SPI1 was not induced during later CFU assay. c Effects of enforced expression of SPI1 on generation of in CD34 + ( left ) and CD34 + CD43 + ( right ) HPCs in H1- GATA2 −/− . Results are presented as mean + SEM of five independent experiments and normalized to H1 group. The data on CD34 + cells generation ( left ) were set as 1 for comparison. The data from five independent experiments were shown as box plot. Asterisks indicate statistical significance determined by t test: * p
    Figure Legend Snippet: Forced expression of SPI1 in H1- GATA2 −/− restores the generation of granulocytes upon OP9 co-culture. a , b Diagram of the strategy of SPI1 rescue experiments. SPI1 linked with a puromycin resistance gene by T2A sequence was controlled by a Dox-inducible promoter in lentiviral-based vectors for Dox-inducible expression of SPI1. The expression of SPI1 was not induced during later CFU assay. c Effects of enforced expression of SPI1 on generation of in CD34 + ( left ) and CD34 + CD43 + ( right ) HPCs in H1- GATA2 −/− . Results are presented as mean + SEM of five independent experiments and normalized to H1 group. The data on CD34 + cells generation ( left ) were set as 1 for comparison. The data from five independent experiments were shown as box plot. Asterisks indicate statistical significance determined by t test: * p

    Techniques Used: Expressing, Co-Culture Assay, Sequencing, Colony-forming Unit Assay

    H1- GATA2 −/− cells restored the potential of granulocyte on OP9-DL1. a Diagram of the strategy of the experiments. H1 or H1- GATA2 −/− ES cells were co-cultured with OP9 for 9 days, then the CD34 + HPCs were harvested and seeded onto OP9 or OP9-DL1 cells for myeloid differentiation. b CD11b and CD14 expression at day 12 of OP9/OP9-DL1-mediated myeloid differentiation were analyzed by FACS. Percentage of total CD11b + myeloid cells or CD11b + CD14 − granulocytes were shown at the left . The right bar charts represent the statistic results of relative generation of CD11b + cells of indicated test ( up ) and the generation of CD11b + CD14 − cells ( down ). The data of H1 and H1- GATA2 −/− from the OP9 co-culture for CD11b + cell generation were set as 1 for comparison. Results indicate mean + SEM of three independent experiments. Asterisks represent statistical significance determined by t test: * p
    Figure Legend Snippet: H1- GATA2 −/− cells restored the potential of granulocyte on OP9-DL1. a Diagram of the strategy of the experiments. H1 or H1- GATA2 −/− ES cells were co-cultured with OP9 for 9 days, then the CD34 + HPCs were harvested and seeded onto OP9 or OP9-DL1 cells for myeloid differentiation. b CD11b and CD14 expression at day 12 of OP9/OP9-DL1-mediated myeloid differentiation were analyzed by FACS. Percentage of total CD11b + myeloid cells or CD11b + CD14 − granulocytes were shown at the left . The right bar charts represent the statistic results of relative generation of CD11b + cells of indicated test ( up ) and the generation of CD11b + CD14 − cells ( down ). The data of H1 and H1- GATA2 −/− from the OP9 co-culture for CD11b + cell generation were set as 1 for comparison. Results indicate mean + SEM of three independent experiments. Asterisks represent statistical significance determined by t test: * p

    Techniques Used: Cell Culture, Expressing, FACS, Co-Culture Assay

    Hematopoietic differentiation of the H1- GATA2 −/− ES cell line. a CFUs of H1 or H1- GATA2 −/− derived CD34 + cells. H1 or H1- GATA2 −/− cells were co-cultured with OP9 cells for 9 days. The CD34 + HPCs were isolated by FACS for CFU generation. Error bars represent mean + SEM of the mean of samples from nine independent experiments. b-d Time course analysis of blood differentiation of H1 and H1- GATA2 −/− cells upon co-culturing with OP9. The expression of surface markers CD34, CD43, and CD31 on H1 or H1- GATA2 −/− cells co-cultured with OP9 for the indicated time was analyzed by FACS ( left and middle panels ). The right panels are box plots from ten independent experiments on the percentage of indicated populations on day 8 of OP9 co-culturing. Asterisks indicate statistical significance determined by t test: *** p
    Figure Legend Snippet: Hematopoietic differentiation of the H1- GATA2 −/− ES cell line. a CFUs of H1 or H1- GATA2 −/− derived CD34 + cells. H1 or H1- GATA2 −/− cells were co-cultured with OP9 cells for 9 days. The CD34 + HPCs were isolated by FACS for CFU generation. Error bars represent mean + SEM of the mean of samples from nine independent experiments. b-d Time course analysis of blood differentiation of H1 and H1- GATA2 −/− cells upon co-culturing with OP9. The expression of surface markers CD34, CD43, and CD31 on H1 or H1- GATA2 −/− cells co-cultured with OP9 for the indicated time was analyzed by FACS ( left and middle panels ). The right panels are box plots from ten independent experiments on the percentage of indicated populations on day 8 of OP9 co-culturing. Asterisks indicate statistical significance determined by t test: *** p

    Techniques Used: Derivative Assay, Cell Culture, Isolation, FACS, Expressing

    Global gene expression analyses of H1 or H1- GATA2 −/− derived HPC and HE. a Hierarchical clustering analysis of RNA-Seq data for indicated samples. Genes with TPMs above 1 and at least threefold change compared with each of any other sample were selected for the analysis. Gene expression values were normalized by Z score and clustered using Clustergram software. The enriched biological functions of the indicated gene group were analyzed by Gene Ontology (GO). HPC: CD34 + CD43 + cells sorted at day 9 of co-culture; HE: CD34 + CD31 + CD43 − cells sorted at day 8 of co-culture. b Paired Pearson correlation analysis of H1 and H1- GATA2 −/− derived HEs ( left ) or HPCs ( right ). R Pearson correlation coefficient, TPM transcripts per million. Selected genes ( red ) are highlighted. c Heat map of selected genes based on TPM value of indicated samples. d Time course analysis of SPI1 expression during the OP9 co-culture by qRT-PCR. Error bars represent SEM of the mean of one single experiment with three replicates, representative of three independent experiments
    Figure Legend Snippet: Global gene expression analyses of H1 or H1- GATA2 −/− derived HPC and HE. a Hierarchical clustering analysis of RNA-Seq data for indicated samples. Genes with TPMs above 1 and at least threefold change compared with each of any other sample were selected for the analysis. Gene expression values were normalized by Z score and clustered using Clustergram software. The enriched biological functions of the indicated gene group were analyzed by Gene Ontology (GO). HPC: CD34 + CD43 + cells sorted at day 9 of co-culture; HE: CD34 + CD31 + CD43 − cells sorted at day 8 of co-culture. b Paired Pearson correlation analysis of H1 and H1- GATA2 −/− derived HEs ( left ) or HPCs ( right ). R Pearson correlation coefficient, TPM transcripts per million. Selected genes ( red ) are highlighted. c Heat map of selected genes based on TPM value of indicated samples. d Time course analysis of SPI1 expression during the OP9 co-culture by qRT-PCR. Error bars represent SEM of the mean of one single experiment with three replicates, representative of three independent experiments

    Techniques Used: Expressing, Derivative Assay, RNA Sequencing Assay, Software, Co-Culture Assay, Quantitative RT-PCR

    Characterization of subtype blood lineages from H1 or H1- GATA2 −/− derived HPCs. a CFU potential cells from H1 or H1- GATA2 −/− were restricted within CD34 + CD43 + subpopulations. EC endothelial cells, MC mesenchymal cells. b Characterization of erythrocytes from H1 or H1- GATA2 −/− . From left to right : phase-contrast photographs of BFU and CFU-E, FACS analysis of CD235a and CD71a expression on H1 and H1- GATA2 −/− derived erythrocytes, and cytospin of H1 and H1- GATA2 −/− derived erythrocytes. c Globin analysis of erythrocytes by RT-qPCR. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. d Analysis of expression of GATA1, GATA2, and GATA3 in H1 or H1- GATA2 −/− derived erythrocytes. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. e Characterization of myeloid cells from H1 or H1- GATA2 −/− . Left : morphologies of indicated CFU colonies; middle : FACS analysis of indicated markers; right : cytospin photographs of indicated colonies. f FACS analysis of CD86 and CD14 expression in H1 and H1- GATA2 −/− derived myeloid CFU. E erythrocyte, G granulocyte, M macrophage, GM G and M, Mix G, E, and M
    Figure Legend Snippet: Characterization of subtype blood lineages from H1 or H1- GATA2 −/− derived HPCs. a CFU potential cells from H1 or H1- GATA2 −/− were restricted within CD34 + CD43 + subpopulations. EC endothelial cells, MC mesenchymal cells. b Characterization of erythrocytes from H1 or H1- GATA2 −/− . From left to right : phase-contrast photographs of BFU and CFU-E, FACS analysis of CD235a and CD71a expression on H1 and H1- GATA2 −/− derived erythrocytes, and cytospin of H1 and H1- GATA2 −/− derived erythrocytes. c Globin analysis of erythrocytes by RT-qPCR. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. d Analysis of expression of GATA1, GATA2, and GATA3 in H1 or H1- GATA2 −/− derived erythrocytes. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. e Characterization of myeloid cells from H1 or H1- GATA2 −/− . Left : morphologies of indicated CFU colonies; middle : FACS analysis of indicated markers; right : cytospin photographs of indicated colonies. f FACS analysis of CD86 and CD14 expression in H1 and H1- GATA2 −/− derived myeloid CFU. E erythrocyte, G granulocyte, M macrophage, GM G and M, Mix G, E, and M

    Techniques Used: Derivative Assay, FACS, Expressing, Quantitative RT-PCR

    23) Product Images from "Azacitidine combined with the selective FLT3 kinase inhibitor crenolanib disrupts stromal protection and inhibits expansion of residual leukemia-initiating cells in FLT3-ITD AML with concurrent epigenetic mutations"

    Article Title: Azacitidine combined with the selective FLT3 kinase inhibitor crenolanib disrupts stromal protection and inhibits expansion of residual leukemia-initiating cells in FLT3-ITD AML with concurrent epigenetic mutations

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21877

    Sensitivity of primary FLT3 -ITD LIC to crenolanib is dependent on concurrent epigenetic gene mutations Experimental design: enriched CD34 + FLT3 -ITD BM cells were cultured on EL08-1D2 stroma and treated on day 1 with DMSO, 10 μM AZA, 100 nM creno or the combination thereof. Cells were harvested after 4 days. Progenitor activity was assessed by short-term colony-forming unit assay (CFU) in methylcellulose. Long-term LIC capacity (long-term culture-derived colony-forming cells, LTC) was assessed after 6 weeks on irradiated (30 Gy) EL08-1D2 cells followed by plating in methylcellulose. Colonies were scored after 14 days using standard criteria. Results are shown as mean CFU (n = 23 ± SEM) and LTC frequencies (n = 17 ± SEM) in relation to DMSO controls (A) . Response to AZA alone regarding co-mutations in NPM1 (CFU n = 23; LTC n =18), DNMT3A (CFU n = 23; LTC n =17), TET2 (CFU n = 23, upper panels; LTC n =17, lower panels) (B) . Response to creno alone regarding co-mutations in NPM1 (CFU n = 23; LTC n =18), DNMT3A (CFU n = 23; LTC n =16), TET2 (CFU n = 23; LTC n =16) (C) .
    Figure Legend Snippet: Sensitivity of primary FLT3 -ITD LIC to crenolanib is dependent on concurrent epigenetic gene mutations Experimental design: enriched CD34 + FLT3 -ITD BM cells were cultured on EL08-1D2 stroma and treated on day 1 with DMSO, 10 μM AZA, 100 nM creno or the combination thereof. Cells were harvested after 4 days. Progenitor activity was assessed by short-term colony-forming unit assay (CFU) in methylcellulose. Long-term LIC capacity (long-term culture-derived colony-forming cells, LTC) was assessed after 6 weeks on irradiated (30 Gy) EL08-1D2 cells followed by plating in methylcellulose. Colonies were scored after 14 days using standard criteria. Results are shown as mean CFU (n = 23 ± SEM) and LTC frequencies (n = 17 ± SEM) in relation to DMSO controls (A) . Response to AZA alone regarding co-mutations in NPM1 (CFU n = 23; LTC n =18), DNMT3A (CFU n = 23; LTC n =17), TET2 (CFU n = 23, upper panels; LTC n =17, lower panels) (B) . Response to creno alone regarding co-mutations in NPM1 (CFU n = 23; LTC n =18), DNMT3A (CFU n = 23; LTC n =16), TET2 (CFU n = 23; LTC n =16) (C) .

    Techniques Used: Cell Culture, Activity Assay, Colony-forming Unit Assay, Derivative Assay, Irradiation

    Detection of FLT3 -ITD and concurrent gene mutations in leukemic stem/progenitor compartments Experimental design (A) . Gating strategy for multiparameter flow cytometric sorting of CD34 + and CD34 - AML BM samples. Blasts (Lin - /CD33 + /CD34 - ), committed progenitors (Lin - /CD33 (+) /CD45 dim /CD34 + CD38 + ), and early stem cell compartments (Lin - /CD33 (+) /CD45 dim /CD34 + CD38 - ) i.e. MLP (CD45RA + ), MPP (CD45RA - CD90 - ) and HSC (CD45RA - CD90 + ). Representative plots for a CD34 + AML (upper panel, Table 1 patient # 2) and CD34 - AML sample (lower panel, Table 1 patient # 7) are shown (B) . gDNA was isolated from sorted compartments and mutations in FLT3 , NPM1 , DNMT3A , TET2 and IDH1/2 genes were detected by targeted resequencing. Variant allele frequencies (VAF, %) of FLT3 -ITD ( C , upper panel) and epigenetic mutations (C, lower panel) in sorted populations are shown. n.a., not available (e.g. population not found).
    Figure Legend Snippet: Detection of FLT3 -ITD and concurrent gene mutations in leukemic stem/progenitor compartments Experimental design (A) . Gating strategy for multiparameter flow cytometric sorting of CD34 + and CD34 - AML BM samples. Blasts (Lin - /CD33 + /CD34 - ), committed progenitors (Lin - /CD33 (+) /CD45 dim /CD34 + CD38 + ), and early stem cell compartments (Lin - /CD33 (+) /CD45 dim /CD34 + CD38 - ) i.e. MLP (CD45RA + ), MPP (CD45RA - CD90 - ) and HSC (CD45RA - CD90 + ). Representative plots for a CD34 + AML (upper panel, Table 1 patient # 2) and CD34 - AML sample (lower panel, Table 1 patient # 7) are shown (B) . gDNA was isolated from sorted compartments and mutations in FLT3 , NPM1 , DNMT3A , TET2 and IDH1/2 genes were detected by targeted resequencing. Variant allele frequencies (VAF, %) of FLT3 -ITD ( C , upper panel) and epigenetic mutations (C, lower panel) in sorted populations are shown. n.a., not available (e.g. population not found).

    Techniques Used: Flow Cytometry, Isolation, Variant Assay

    24) Product Images from "Clinical and Molecular Heterogeneity of RTEL1 Deficiency"

    Article Title: Clinical and Molecular Heterogeneity of RTEL1 Deficiency

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00449

    Impaired proliferative capacity of B-cell progenitors in vivo and CD34 + cells in vitro . (A) Normal ontogeny of B-cells in bone marrow (BM). B Prec, B-cell precursors; CLP, common lymphoid progenitor; HSC, hematopoietic stem cells; ImmB, Immature B-cells. (B) B-cell differentiation is markedly impaired in BM of P1 during severe adenovirus infection. CLP/ProB subsets are greatly reduced while B cell precursors are missing. (C) Between infectious intervals, all populations recover. (D,E) Total and CD34 + selected HSC from P3 and P5 fail to expand in non-differentiating culture. Ctrl, healthy control.
    Figure Legend Snippet: Impaired proliferative capacity of B-cell progenitors in vivo and CD34 + cells in vitro . (A) Normal ontogeny of B-cells in bone marrow (BM). B Prec, B-cell precursors; CLP, common lymphoid progenitor; HSC, hematopoietic stem cells; ImmB, Immature B-cells. (B) B-cell differentiation is markedly impaired in BM of P1 during severe adenovirus infection. CLP/ProB subsets are greatly reduced while B cell precursors are missing. (C) Between infectious intervals, all populations recover. (D,E) Total and CD34 + selected HSC from P3 and P5 fail to expand in non-differentiating culture. Ctrl, healthy control.

    Techniques Used: In Vivo, In Vitro, Cell Differentiation, Infection

    25) Product Images from "Genotyping of Transcriptomes links somatic mutations and cell identity"

    Article Title: Genotyping of Transcriptomes links somatic mutations and cell identity

    Journal: Nature

    doi: 10.1038/s41586-019-1367-0

    CALR mutation effects on hematopoietic progenitor cells from MF patients. a, t-SNE projection of CD34 + cells from MF patients showing cluster assignment and b, genotyping data from GoT. c, ). Bar graphs represent aggregate analysis of MF01-MF04 showing mean ± SD of 100 downsampling iterations to 1 genotyping UMI; gray points represent mean of 100 down-sampling iterations for each sample. d, t-SNE projection of the CD34 + ). e, ). P ).
    Figure Legend Snippet: CALR mutation effects on hematopoietic progenitor cells from MF patients. a, t-SNE projection of CD34 + cells from MF patients showing cluster assignment and b, genotyping data from GoT. c, ). Bar graphs represent aggregate analysis of MF01-MF04 showing mean ± SD of 100 downsampling iterations to 1 genotyping UMI; gray points represent mean of 100 down-sampling iterations for each sample. d, t-SNE projection of the CD34 + ). e, ). P ).

    Techniques Used: Mutagenesis, Sampling

    CALR -mutated hematopoietic progenitor cells from myelofibrosis show upregulation of the IRE1-unfolded protein response. a, t-SNE projection of CD34 + , n = 11,093). b, t-SNE projection of CD34 + (left, n = 11,093). Pseudotime comparison between wildtype (n = 2221) and mutant (n = 7483) cells. P ). c, Cell cycle module score comparison between wildtype and mutant cells in MF patients (Wilcoxon rank-sum test, two-sided). d, Ratio of TGFβ signaling pathway gene expression of mutant and wildtype MkPs. One mutant and one wildtype MkPs were randomly sampled for each round of analysis (n = 100 iterations; Wilcoxon-rank sum test, two-sided). e, Differentially expressed genes between wildtype MkPs with high cell cycle expression (n = 220) vs. wildtype MkPs with low cell cycle expression (n = 110) common across patient samples MF02-MF04. P -values were combined using Fisher combine test with Benjamini-Hochberg adjustments. Weighted average of log 2 ).
    Figure Legend Snippet: CALR -mutated hematopoietic progenitor cells from myelofibrosis show upregulation of the IRE1-unfolded protein response. a, t-SNE projection of CD34 + , n = 11,093). b, t-SNE projection of CD34 + (left, n = 11,093). Pseudotime comparison between wildtype (n = 2221) and mutant (n = 7483) cells. P ). c, Cell cycle module score comparison between wildtype and mutant cells in MF patients (Wilcoxon rank-sum test, two-sided). d, Ratio of TGFβ signaling pathway gene expression of mutant and wildtype MkPs. One mutant and one wildtype MkPs were randomly sampled for each round of analysis (n = 100 iterations; Wilcoxon-rank sum test, two-sided). e, Differentially expressed genes between wildtype MkPs with high cell cycle expression (n = 220) vs. wildtype MkPs with low cell cycle expression (n = 110) common across patient samples MF02-MF04. P -values were combined using Fisher combine test with Benjamini-Hochberg adjustments. Weighted average of log 2 ).

    Techniques Used: Mutagenesis, Expressing

    Integration of ET patient samples and progenitor subset assignment. a, t-SNE projection of CD34 + ). b, Heatmap of top ten differentially expressed genes for clusters; lineage-specific genes from Velten et al. ). c, Representative lineage-specific genes projected on t-SNE representation of CD34 + cells from ET patient samples. d, t-SNE projection of CD34 + showing progenitor subset assignments as determined after clustering the cells using the Seurat package. e, Genotyping data from GoT are projected onto the t-SNE generated after the scVI analysis of progenitor cells from ET01-ET05. Cells without any GoT data are labeled NA (not assignable). WT, wildtype; MUT, mutant; HSPC, hematopoietic stem progenitor cells; IMP, immature myeloid progenitors; NP, neutrophil progenitors; M/D, monocyte-dendritic cell progenitors; E/B/M, eosinophil, basophil, mast cell progenitors; MEP, megakaryocytic-erythroid progenitors; MkP, megakaryocytic progenitors; EP, erythroid progenitors; PreB, precursor B-cells.
    Figure Legend Snippet: Integration of ET patient samples and progenitor subset assignment. a, t-SNE projection of CD34 + ). b, Heatmap of top ten differentially expressed genes for clusters; lineage-specific genes from Velten et al. ). c, Representative lineage-specific genes projected on t-SNE representation of CD34 + cells from ET patient samples. d, t-SNE projection of CD34 + showing progenitor subset assignments as determined after clustering the cells using the Seurat package. e, Genotyping data from GoT are projected onto the t-SNE generated after the scVI analysis of progenitor cells from ET01-ET05. Cells without any GoT data are labeled NA (not assignable). WT, wildtype; MUT, mutant; HSPC, hematopoietic stem progenitor cells; IMP, immature myeloid progenitors; NP, neutrophil progenitors; M/D, monocyte-dendritic cell progenitors; E/B/M, eosinophil, basophil, mast cell progenitors; MEP, megakaryocytic-erythroid progenitors; MkP, megakaryocytic progenitors; EP, erythroid progenitors; PreB, precursor B-cells.

    Techniques Used: Generated, Labeling, Mutagenesis

    Results of GoT analysis is robust to various amplicon UMI thresholds and linear modeling. a, for sample size). b, ). c, Pseudotime comparison between mutant and wildtype cells with UMI threshold of 1 (Extended Data Figure 5b) with statistical test using a generalized linear model including mutation status and total number of amplicon UMIs per cell. d, Across 100 iterations, the genotyping amplicon UMIs were downsampled to one per cell and mutant cell frequency was determined for MkPs or precursor B-cells (PreB). This frequency was then divided by the total mutant cell frequency across all progenitor subsets for each of the 100 iterations. Mean ± standard deviation (SD) after n = 100 down-sampling iterations (Wilcoxon rank-sum test, two sided). ET samples with at least 20 cells in each cluster were analyzed. e, Variant allele fraction of CALR mutation in CD34 + , CD38 − (left), CD34 + , CD38 + (middle) and CD34 + , CD10 + (right) FACS-sorted peripheral blood cells from patients with ET determined by droplet digital (dd) PCR.
    Figure Legend Snippet: Results of GoT analysis is robust to various amplicon UMI thresholds and linear modeling. a, for sample size). b, ). c, Pseudotime comparison between mutant and wildtype cells with UMI threshold of 1 (Extended Data Figure 5b) with statistical test using a generalized linear model including mutation status and total number of amplicon UMIs per cell. d, Across 100 iterations, the genotyping amplicon UMIs were downsampled to one per cell and mutant cell frequency was determined for MkPs or precursor B-cells (PreB). This frequency was then divided by the total mutant cell frequency across all progenitor subsets for each of the 100 iterations. Mean ± standard deviation (SD) after n = 100 down-sampling iterations (Wilcoxon rank-sum test, two sided). ET samples with at least 20 cells in each cluster were analyzed. e, Variant allele fraction of CALR mutation in CD34 + , CD38 − (left), CD34 + , CD38 + (middle) and CD34 + , CD10 + (right) FACS-sorted peripheral blood cells from patients with ET determined by droplet digital (dd) PCR.

    Techniques Used: Amplification, Mutagenesis, Standard Deviation, Sampling, Variant Assay, FACS, Polymerase Chain Reaction

    GoT captures genotyping information of single cells through cDNA. a, Percentage of cells by number of UMIs with CALR mutation locus capture in standard 10x data (left) and GoT data (right) (see Extended Data Fig. 3c for cell number in each sample). b, Number of UMIs per cell of CALR transcript from standard 10x data (left, blue shade) or targeted CALR locus from standard 10x or GoT (pink shade, see Extended Data Fig. 3c for cell number in each sample). c, Summary of clinical, pathologic, and GoT data from patients with CALR -mutated myeloproliferative neoplasms. BM, bone marrow; PB, peripheral blood. d, Number of genes per cell (left) and number of UMIs per cell (right) from published standard 10x data of healthy control CD34 + cells and 10x data from 3’ v2 chemistry of CD34 + cells from patient samples that underwent concurrent GoT, after random down-sampling of the reads from each sample to 50 million reads x3 iterations, showing that extra cycle of PCR and portioning a small aliquot from the 10x cDNA library for GoT using 3’ v2 chemistry does not compromise scRNA-seq data.
    Figure Legend Snippet: GoT captures genotyping information of single cells through cDNA. a, Percentage of cells by number of UMIs with CALR mutation locus capture in standard 10x data (left) and GoT data (right) (see Extended Data Fig. 3c for cell number in each sample). b, Number of UMIs per cell of CALR transcript from standard 10x data (left, blue shade) or targeted CALR locus from standard 10x or GoT (pink shade, see Extended Data Fig. 3c for cell number in each sample). c, Summary of clinical, pathologic, and GoT data from patients with CALR -mutated myeloproliferative neoplasms. BM, bone marrow; PB, peripheral blood. d, Number of genes per cell (left) and number of UMIs per cell (right) from published standard 10x data of healthy control CD34 + cells and 10x data from 3’ v2 chemistry of CD34 + cells from patient samples that underwent concurrent GoT, after random down-sampling of the reads from each sample to 50 million reads x3 iterations, showing that extra cycle of PCR and portioning a small aliquot from the 10x cDNA library for GoT using 3’ v2 chemistry does not compromise scRNA-seq data.

    Techniques Used: Mutagenesis, Sampling, Polymerase Chain Reaction, cDNA Library Assay

    GoT dissects subclonal identity through multiplexing and targets loci distant from transcript ends via circularization. a, Schematic of clonal evolution of neoplastic cells from MF05 (top-left). t-SNE projections of CD34 + cells with cluster assignments (top-right) and with with GoT data for each variant (bottom). b, Cell cycle score in subclonal MEP populations (n = 28 single mutant, 109 double mutant, 293 triple mutant cells). c, Schematic of circularization GoT. d, t-SNE projection of MF05 CD34 + cells GoT data for SF3B1 from circularization and linear GoT. e, UMI per cell of SF3B1 gene (blue shade) or targeted SF3B1 locus (pink shade) from 10x, linear GoT sequenced on Illumina, circularization GoT, and linear GoT sequenced with ONT (n = 8475 cells). f, Mixing study with human JAK2 wildtype cDNA from TF-1 and homozygous JAK2 V617F cDNA from HEL. Frequency of reads (wildtype, V617F or not assignable [NA]) assigned to TF-1 or HEL cell barcodes (CB). g, t-SNE projection of CD34 + cells from patient with JAK2 V617F ET showing cluster assignment (left) and genotyping information (right) based on GoT data. h, ). Mean ± SD of n = 100 down-sampling iterations. i, Density plots of HSPCs along lineage priming modules (n = 17 wildtype vs. 15 mutant cells). P -values for b , h , i from Wilcoxon rank-sum test, two-sided.
    Figure Legend Snippet: GoT dissects subclonal identity through multiplexing and targets loci distant from transcript ends via circularization. a, Schematic of clonal evolution of neoplastic cells from MF05 (top-left). t-SNE projections of CD34 + cells with cluster assignments (top-right) and with with GoT data for each variant (bottom). b, Cell cycle score in subclonal MEP populations (n = 28 single mutant, 109 double mutant, 293 triple mutant cells). c, Schematic of circularization GoT. d, t-SNE projection of MF05 CD34 + cells GoT data for SF3B1 from circularization and linear GoT. e, UMI per cell of SF3B1 gene (blue shade) or targeted SF3B1 locus (pink shade) from 10x, linear GoT sequenced on Illumina, circularization GoT, and linear GoT sequenced with ONT (n = 8475 cells). f, Mixing study with human JAK2 wildtype cDNA from TF-1 and homozygous JAK2 V617F cDNA from HEL. Frequency of reads (wildtype, V617F or not assignable [NA]) assigned to TF-1 or HEL cell barcodes (CB). g, t-SNE projection of CD34 + cells from patient with JAK2 V617F ET showing cluster assignment (left) and genotyping information (right) based on GoT data. h, ). Mean ± SD of n = 100 down-sampling iterations. i, Density plots of HSPCs along lineage priming modules (n = 17 wildtype vs. 15 mutant cells). P -values for b , h , i from Wilcoxon rank-sum test, two-sided.

    Techniques Used: Multiplexing, Variant Assay, Mutagenesis, Sampling

    26) Product Images from "GM-CSF Promotes the Expansion and Differentiation of Cord Blood Myeloid-Derived Suppressor Cells, Which Attenuate Xenogeneic Graft-vs.-Host Disease"

    Article Title: GM-CSF Promotes the Expansion and Differentiation of Cord Blood Myeloid-Derived Suppressor Cells, Which Attenuate Xenogeneic Graft-vs.-Host Disease

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00183

    Expansion of CB CD34 + cells according to cytokine combinations and differentiation into MDSCs. (A) The scheme of cell expansion or differentiation from CB CD34 + cells. (B) Fold expansion by different cytokine combinations. The culture medium was replaced with each cytokine combinations every week. (C) The FITC conjugated anti-human CD33 were compensated with compensation bead to avoid spillover with PE conjugated anti-human CD11b. (D) Expression of CD11b + CD33 + . The cells were stained with fluorochrome conjugated anti-human CD33 (FITC) and aniti-human CD11b (PE) every week. (E) The CD34 + cells were cultured with human GM-CSF (100 ng/ml) and human SCF (50 ng/ml) for 3 weeks and the cells were stained like (D) . The stained cells were sorted by FACS Aria and the sorted cells (CD11b + CD33b + vs. CD11b − CD33 + ) were cultured with human GM-CSF (100ng/ml) and human SCF (50ng/ml) for a further 1 week. After one week, the sorted cells were stained like (D) and analyzed by flow cytometry. (F) Expression of HLA-DR, CD14 and CD15. The cells were stained with efluor450-conjugated anti-human HLA-DR, PE-Cy7 anti-human CD14 and APC anti-human CD15 from 3 to 6 weeks, respectively and analyzed by flow cytometry. These experiments were reproduced in 10 individuals (from B to D and F ) and 6 individuals (E) .
    Figure Legend Snippet: Expansion of CB CD34 + cells according to cytokine combinations and differentiation into MDSCs. (A) The scheme of cell expansion or differentiation from CB CD34 + cells. (B) Fold expansion by different cytokine combinations. The culture medium was replaced with each cytokine combinations every week. (C) The FITC conjugated anti-human CD33 were compensated with compensation bead to avoid spillover with PE conjugated anti-human CD11b. (D) Expression of CD11b + CD33 + . The cells were stained with fluorochrome conjugated anti-human CD33 (FITC) and aniti-human CD11b (PE) every week. (E) The CD34 + cells were cultured with human GM-CSF (100 ng/ml) and human SCF (50 ng/ml) for 3 weeks and the cells were stained like (D) . The stained cells were sorted by FACS Aria and the sorted cells (CD11b + CD33b + vs. CD11b − CD33 + ) were cultured with human GM-CSF (100ng/ml) and human SCF (50ng/ml) for a further 1 week. After one week, the sorted cells were stained like (D) and analyzed by flow cytometry. (F) Expression of HLA-DR, CD14 and CD15. The cells were stained with efluor450-conjugated anti-human HLA-DR, PE-Cy7 anti-human CD14 and APC anti-human CD15 from 3 to 6 weeks, respectively and analyzed by flow cytometry. These experiments were reproduced in 10 individuals (from B to D and F ) and 6 individuals (E) .

    Techniques Used: Expressing, Staining, Cell Culture, FACS, Flow Cytometry, Cytometry

    27) Product Images from "Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells "

    Article Title: Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells

    Journal: The Journal of Experimental Medicine

    doi:

    Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).
    Figure Legend Snippet: Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).

    Techniques Used: Chemotaxis Assay, Derivative Assay

    RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.
    Figure Legend Snippet: RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Molecular Weight, Derivative Assay

    28) Product Images from "Knockdown of Hspa9, a del(5q31.2) gene, results in a decrease in hematopoietic progenitors in mice"

    Article Title: Knockdown of Hspa9, a del(5q31.2) gene, results in a decrease in hematopoietic progenitors in mice

    Journal: Blood

    doi: 10.1182/blood-2010-06-293167

    Knockdown of HSPA9 in human hematopoietic progenitor cells alters their erythroid differentiation. (A) Representative immunoblot of HSPA9 protein in CD34 + cells transduced with shRNAs targeting control genes (empty or luciferase) or HSPA9 after 7 days
    Figure Legend Snippet: Knockdown of HSPA9 in human hematopoietic progenitor cells alters their erythroid differentiation. (A) Representative immunoblot of HSPA9 protein in CD34 + cells transduced with shRNAs targeting control genes (empty or luciferase) or HSPA9 after 7 days

    Techniques Used: Transduction, Luciferase

    29) Product Images from "An RNA editing fingerprint of cancer stem cell reprogramming"

    Article Title: An RNA editing fingerprint of cancer stem cell reprogramming

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-014-0370-3

    Validation and quantification of RNA editing activity in primary bone marrow-derived hematopoietic stem and progenitor cells transduced with lentiviral-ADAR1. CD34-selected cells from normal bone marrow (BM) samples (n = 3, average donor age = 64.3 ± 2.9 years old) were transduced with lentiviral (lenti)-ADAR1 or vector (ORF) control. After 4 days of culture, cells were lysed and processed for qRT-PCR and RESSq-PCR analysis. (A,B) Relative expression of lentivirus-derived (a) and total (b) ADAR1 levels in transduced BM samples (n = 3) showing increased human ADAR1 expression in ADAR1-transduced samples, with higher levels of total ADAR1 overexpression achieved in samples BM-410 and BM-416. (C,D) Representative Sanger sequencing analysis of high-fidelity PCR products amplified with primers flanking the APOBEC3D editing site showing increased G(I) peak in lenti-ADAR1 transduced cells that displayed robust ADAR1 expression (BM-410, C). (E,F) Quantification of sequencing peak height ratios and corresponding RESSq-PCR analysis in lenti-ORF and lenti-ADAR1 transduced BM samples.
    Figure Legend Snippet: Validation and quantification of RNA editing activity in primary bone marrow-derived hematopoietic stem and progenitor cells transduced with lentiviral-ADAR1. CD34-selected cells from normal bone marrow (BM) samples (n = 3, average donor age = 64.3 ± 2.9 years old) were transduced with lentiviral (lenti)-ADAR1 or vector (ORF) control. After 4 days of culture, cells were lysed and processed for qRT-PCR and RESSq-PCR analysis. (A,B) Relative expression of lentivirus-derived (a) and total (b) ADAR1 levels in transduced BM samples (n = 3) showing increased human ADAR1 expression in ADAR1-transduced samples, with higher levels of total ADAR1 overexpression achieved in samples BM-410 and BM-416. (C,D) Representative Sanger sequencing analysis of high-fidelity PCR products amplified with primers flanking the APOBEC3D editing site showing increased G(I) peak in lenti-ADAR1 transduced cells that displayed robust ADAR1 expression (BM-410, C). (E,F) Quantification of sequencing peak height ratios and corresponding RESSq-PCR analysis in lenti-ORF and lenti-ADAR1 transduced BM samples.

    Techniques Used: Activity Assay, Derivative Assay, Transduction, Plasmid Preparation, Quantitative RT-PCR, Polymerase Chain Reaction, Expressing, Over Expression, Sequencing, Amplification

    In vitro humanized stromal co-culture model and RESSq-PCR analysis of primary CP CML cells transduced with lentiviral-ADAR1. (A) Schematic diagram of humanized bone marrow stromal co-culture assay. CD34-selected hematopoietic stem and progenitor cells (HSPC) isolated from patients with CP CML were transduced with lenti-ADAR1 or ORF control. After 3 days of culture, cells were transferred to SL/M2 mouse bone marrow stromal monolayers for co-culture and subsequent RESSq-PCR analysis. (B,C) Increased total ADAR1 (B) and lenti-ADAR1 (C) expression in transduced CP CML samples (n = 3). (D) RESSq-PCR analysis showing increased APOBEC3D RNA editing in lenti-ADAR1 transduced cells from patients with CP CML that harbored high ADAR1 expression after transduction. Horizontal dashed lines represent comparative RNA editing activity in K562-ADAR1 and K562-ORF cells.
    Figure Legend Snippet: In vitro humanized stromal co-culture model and RESSq-PCR analysis of primary CP CML cells transduced with lentiviral-ADAR1. (A) Schematic diagram of humanized bone marrow stromal co-culture assay. CD34-selected hematopoietic stem and progenitor cells (HSPC) isolated from patients with CP CML were transduced with lenti-ADAR1 or ORF control. After 3 days of culture, cells were transferred to SL/M2 mouse bone marrow stromal monolayers for co-culture and subsequent RESSq-PCR analysis. (B,C) Increased total ADAR1 (B) and lenti-ADAR1 (C) expression in transduced CP CML samples (n = 3). (D) RESSq-PCR analysis showing increased APOBEC3D RNA editing in lenti-ADAR1 transduced cells from patients with CP CML that harbored high ADAR1 expression after transduction. Horizontal dashed lines represent comparative RNA editing activity in K562-ADAR1 and K562-ORF cells.

    Techniques Used: In Vitro, Co-Culture Assay, Polymerase Chain Reaction, Transduction, Co-culture Assay, Isolation, Expressing, Activity Assay

    Detection of increased RNA editing activity by RESSq-PCR analysis of primary chronic phase versus blast crisis CML progenitors. RNA extracted from FACS-purified CD34 + CD38 + Lin - primary CML progenitors was analyzed by RESSq-PCR to validate the RNA editing fingerprint of leukemic progression. (A) RESSq-PCR analysis detecting increased RNA editing in APOBEC3D in purified BC CML LSC versus CP progenitors. (B) RESSq-PCR analysis detecting increased RNA editing in AZIN1 in purified BC CML LSC versus CP progenitors. Horizontal dashed lines represent comparative RNA editing activity in K562-ADAR1 and K562-ORF cells.
    Figure Legend Snippet: Detection of increased RNA editing activity by RESSq-PCR analysis of primary chronic phase versus blast crisis CML progenitors. RNA extracted from FACS-purified CD34 + CD38 + Lin - primary CML progenitors was analyzed by RESSq-PCR to validate the RNA editing fingerprint of leukemic progression. (A) RESSq-PCR analysis detecting increased RNA editing in APOBEC3D in purified BC CML LSC versus CP progenitors. (B) RESSq-PCR analysis detecting increased RNA editing in AZIN1 in purified BC CML LSC versus CP progenitors. Horizontal dashed lines represent comparative RNA editing activity in K562-ADAR1 and K562-ORF cells.

    Techniques Used: Activity Assay, Polymerase Chain Reaction, FACS, Purification

    30) Product Images from "Clinical and Molecular Heterogeneity of RTEL1 Deficiency"

    Article Title: Clinical and Molecular Heterogeneity of RTEL1 Deficiency

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00449

    Impaired proliferative capacity of B-cell progenitors in vivo and CD34 + cells in vitro . (A) Normal ontogeny of B-cells in bone marrow (BM). B Prec, B-cell precursors; CLP, common lymphoid progenitor; HSC, hematopoietic stem cells; ImmB, Immature B-cells. (B) B-cell differentiation is markedly impaired in BM of P1 during severe adenovirus infection. CLP/ProB subsets are greatly reduced while B cell precursors are missing. (C) Between infectious intervals, all populations recover. (D,E) Total and CD34 + selected HSC from P3 and P5 fail to expand in non-differentiating culture. Ctrl, healthy control.
    Figure Legend Snippet: Impaired proliferative capacity of B-cell progenitors in vivo and CD34 + cells in vitro . (A) Normal ontogeny of B-cells in bone marrow (BM). B Prec, B-cell precursors; CLP, common lymphoid progenitor; HSC, hematopoietic stem cells; ImmB, Immature B-cells. (B) B-cell differentiation is markedly impaired in BM of P1 during severe adenovirus infection. CLP/ProB subsets are greatly reduced while B cell precursors are missing. (C) Between infectious intervals, all populations recover. (D,E) Total and CD34 + selected HSC from P3 and P5 fail to expand in non-differentiating culture. Ctrl, healthy control.

    Techniques Used: In Vivo, In Vitro, Cell Differentiation, Infection

    31) Product Images from "Osteogenic Differentiation of Human Amniotic Fluid Mesenchymal Stem Cells Is Determined by Epigenetic Changes"

    Article Title: Osteogenic Differentiation of Human Amniotic Fluid Mesenchymal Stem Cells Is Determined by Epigenetic Changes

    Journal: Stem Cells International

    doi: 10.1155/2016/6465307

    Characterization of AF-MSCs. (a) Representative image of spindle-shaped AF-MSCs. Scale bar = 400 μ m. (b) Expression of cell surface markers CD45, CD34, CD105, and CD90 measured by using FACS and FITC or PE labeled antibodies. The data was represented as mean with standard deviation ( n = 3). (c) Expression of stem cells pluripotency markers Oct4 , Sox2 , Nanog , and Rex1 as determined by RT-qPCR. The data were normalized to GAPDH and presented as mean ± SD ( n = 3). (d) AF-MSCs after induction of osteogenic differentiation for 15 days, stained with Alizarin Red. Calcified extracellular matrix deposition is colored red proving osteogenic differentiation.
    Figure Legend Snippet: Characterization of AF-MSCs. (a) Representative image of spindle-shaped AF-MSCs. Scale bar = 400 μ m. (b) Expression of cell surface markers CD45, CD34, CD105, and CD90 measured by using FACS and FITC or PE labeled antibodies. The data was represented as mean with standard deviation ( n = 3). (c) Expression of stem cells pluripotency markers Oct4 , Sox2 , Nanog , and Rex1 as determined by RT-qPCR. The data were normalized to GAPDH and presented as mean ± SD ( n = 3). (d) AF-MSCs after induction of osteogenic differentiation for 15 days, stained with Alizarin Red. Calcified extracellular matrix deposition is colored red proving osteogenic differentiation.

    Techniques Used: Expressing, FACS, Labeling, Standard Deviation, Quantitative RT-PCR, Staining

    Related Articles

    Selection:

    Article Title: Effect of increased HoxB4 on human megakaryocytic development
    Article Snippet: Light–density cells were isolated from citrated cord blood using discontinuous density centrifugation over Ficoll–Paque Plus (GE Healthcare BioSciences, Uppsala, Sweden). .. CD34-positive selection was conducted using a MACS Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Anaheim, CA, USA). .. To induce megakaryocytic differentiation, the CD34 positive cells were cultured in IMDM medium with 10% BIT 9500 Serum Substitute (Stemcell Technologies), 50ng/ml SCF, 50ng/ml TPO, 50ng/ml Flt, 10ng/ml IL6, 10ng/ml IL3 and 10ng/ml IL11 (All cytokines from R & D Systems Inc., Minneapolis, MN).

    Article Title: Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform
    Article Snippet: Procedures for the use of UCB for this study were reviewed and approved by the medical ethics committee of Hannover Medical School. .. CB mononuclear cells were isolated using Ficoll density centrifugation and CD34 selection was performed using a CD34 microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. .. Purity of CD34+ cells was higher than 95% as determined by postenrichment flow cytometric analysis.

    Magnetic Cell Separation:

    Article Title: Effect of increased HoxB4 on human megakaryocytic development
    Article Snippet: Light–density cells were isolated from citrated cord blood using discontinuous density centrifugation over Ficoll–Paque Plus (GE Healthcare BioSciences, Uppsala, Sweden). .. CD34-positive selection was conducted using a MACS Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Anaheim, CA, USA). .. To induce megakaryocytic differentiation, the CD34 positive cells were cultured in IMDM medium with 10% BIT 9500 Serum Substitute (Stemcell Technologies), 50ng/ml SCF, 50ng/ml TPO, 50ng/ml Flt, 10ng/ml IL6, 10ng/ml IL3 and 10ng/ml IL11 (All cytokines from R & D Systems Inc., Minneapolis, MN).

    Article Title: A Virally Encoded DeSUMOylase Activity Is Required for Cytomegalovirus Reactivation from Latency
    Article Snippet: Two washing steps in 2× saline sodium citrate buffer (5 min, 60°C) were followed by a PBS wash at room temperature and then staining as before to detect PML localization. .. Primary CD34+ hematopoietic cells were isolated from apheresis blood packs harvested from cranulocyte-colony-stimulating factor-mobilized donors using CD34+ magnetic activated cell sorting (MACS) separation (Miltenyi Biotec). .. Alternatively, CD34+ cells isolated from granulocyte-colony-stimulating factor (G-CSF)-mobilized healthy donors were purchased for use in some experiments (Lonza).The inhibition of isopeptidase activity was achieved using G5 (1 μM unless stated otherwise; Calbiochem) by addition of the chemical directly to the culture media 8 hr post-transfection or 3 hr prior to the addition of LPS in reactivation studies.

    Cell Isolation:

    Article Title: Effect of increased HoxB4 on human megakaryocytic development
    Article Snippet: Light–density cells were isolated from citrated cord blood using discontinuous density centrifugation over Ficoll–Paque Plus (GE Healthcare BioSciences, Uppsala, Sweden). .. CD34-positive selection was conducted using a MACS Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Anaheim, CA, USA). .. To induce megakaryocytic differentiation, the CD34 positive cells were cultured in IMDM medium with 10% BIT 9500 Serum Substitute (Stemcell Technologies), 50ng/ml SCF, 50ng/ml TPO, 50ng/ml Flt, 10ng/ml IL6, 10ng/ml IL3 and 10ng/ml IL11 (All cytokines from R & D Systems Inc., Minneapolis, MN).

    Isolation:

    Article Title: Targeting mitochondrial respiration selectively sensitizes pediatric acute lymphoblastic leukemia cell lines and patient samples to standard chemotherapy
    Article Snippet: .. ALL CD34 cells and ALL lymphocytes were isolated from bone marrow or peripheral blood of ALL patients using CD34 MicroBead kit and HLA B/T Cell Isolation Kit (Miltenyi Biotec, Germany), respectively. ..

    Article Title: A Virally Encoded DeSUMOylase Activity Is Required for Cytomegalovirus Reactivation from Latency
    Article Snippet: Two washing steps in 2× saline sodium citrate buffer (5 min, 60°C) were followed by a PBS wash at room temperature and then staining as before to detect PML localization. .. Primary CD34+ hematopoietic cells were isolated from apheresis blood packs harvested from cranulocyte-colony-stimulating factor-mobilized donors using CD34+ magnetic activated cell sorting (MACS) separation (Miltenyi Biotec). .. Alternatively, CD34+ cells isolated from granulocyte-colony-stimulating factor (G-CSF)-mobilized healthy donors were purchased for use in some experiments (Lonza).The inhibition of isopeptidase activity was achieved using G5 (1 μM unless stated otherwise; Calbiochem) by addition of the chemical directly to the culture media 8 hr post-transfection or 3 hr prior to the addition of LPS in reactivation studies.

    Article Title: Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform
    Article Snippet: Procedures for the use of UCB for this study were reviewed and approved by the medical ethics committee of Hannover Medical School. .. CB mononuclear cells were isolated using Ficoll density centrifugation and CD34 selection was performed using a CD34 microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. .. Purity of CD34+ cells was higher than 95% as determined by postenrichment flow cytometric analysis.

    Purification:

    Article Title: Variable Behavior of iPSCs Derived from CML Patients for Response to TKI and Hematopoietic Differentiation
    Article Snippet: Briefly, mononuclear cells were isolated by Ficoll gradient. .. CD34+ cells were purified according to the manufacturer's instructions (Miltenyi Biotech) and purity was analyzed by flow cytometry using phycoerythrin-conjugated anti-CD34 antibody (Becton Dickinson). .. Cryopreserved CD34+ cells were thawed and cultured 2 days in expansion medium consisting in Stem Span SFEM (Stem cell Technologies, Grenoble, France) supplemented with Flt3-L (50 ng/mL), SCF (50 ng/mL) and human TPO (50 ng/mL) (all from Peprotech, Rocky Hill, NJ, USA). iPSCs generation were obtained by transduction of CD34+ cells with the two excisable SIN-lentivectors OSK1 and Mshp53 (flanked by LoxP sites) at a multiplicity of infection (MOI) of 100 .

    Flow Cytometry:

    Article Title: Variable Behavior of iPSCs Derived from CML Patients for Response to TKI and Hematopoietic Differentiation
    Article Snippet: Briefly, mononuclear cells were isolated by Ficoll gradient. .. CD34+ cells were purified according to the manufacturer's instructions (Miltenyi Biotech) and purity was analyzed by flow cytometry using phycoerythrin-conjugated anti-CD34 antibody (Becton Dickinson). .. Cryopreserved CD34+ cells were thawed and cultured 2 days in expansion medium consisting in Stem Span SFEM (Stem cell Technologies, Grenoble, France) supplemented with Flt3-L (50 ng/mL), SCF (50 ng/mL) and human TPO (50 ng/mL) (all from Peprotech, Rocky Hill, NJ, USA). iPSCs generation were obtained by transduction of CD34+ cells with the two excisable SIN-lentivectors OSK1 and Mshp53 (flanked by LoxP sites) at a multiplicity of infection (MOI) of 100 .

    Cytometry:

    Article Title: Variable Behavior of iPSCs Derived from CML Patients for Response to TKI and Hematopoietic Differentiation
    Article Snippet: Briefly, mononuclear cells were isolated by Ficoll gradient. .. CD34+ cells were purified according to the manufacturer's instructions (Miltenyi Biotech) and purity was analyzed by flow cytometry using phycoerythrin-conjugated anti-CD34 antibody (Becton Dickinson). .. Cryopreserved CD34+ cells were thawed and cultured 2 days in expansion medium consisting in Stem Span SFEM (Stem cell Technologies, Grenoble, France) supplemented with Flt3-L (50 ng/mL), SCF (50 ng/mL) and human TPO (50 ng/mL) (all from Peprotech, Rocky Hill, NJ, USA). iPSCs generation were obtained by transduction of CD34+ cells with the two excisable SIN-lentivectors OSK1 and Mshp53 (flanked by LoxP sites) at a multiplicity of infection (MOI) of 100 .

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells
    Article Snippet: .. RT-PCR on RNA extracted from distinct human leukocyte populations indicated that DCCR2 mRNA expression was restricted to T cells, CD34+ DCs, and lung DCs but not on peripheral blood monocyte–derived DCs (Fig. ). .. Full-length DCCR2 was subsequently cloned from human lung DCs by RT-PCR using specific primers based on the full-length sequence given in the EMBL/GenBank/DDBJ database under accession number U48494 .

    Expressing:

    Article Title: Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells
    Article Snippet: .. RT-PCR on RNA extracted from distinct human leukocyte populations indicated that DCCR2 mRNA expression was restricted to T cells, CD34+ DCs, and lung DCs but not on peripheral blood monocyte–derived DCs (Fig. ). .. Full-length DCCR2 was subsequently cloned from human lung DCs by RT-PCR using specific primers based on the full-length sequence given in the EMBL/GenBank/DDBJ database under accession number U48494 .

    other:

    Article Title: Reprogramming mechanisms influence the maturation of hematopoietic progenitors from human pluripotent stem cells
    Article Snippet: Fuhrken PG, et al. Gene Ontology-driven transcriptional analysis of CD34+cell-initiated megakaryocytic cultures identifies new transcriptional regulators of megakaryopoiesis.

    Gradient Centrifugation:

    Article Title: Effect of intravenous coadministration of human stroma cell lines on engraftment of long-term repopulating clonal myelodysplastic syndrome cells in immunodeficient mice
    Article Snippet: .. Bone marrow mononuclear cells and PB cells were separated by Ficoll–Hypaque gradient centrifugation and suspended in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum until use, or were subjected to magnetic-activated cell sorting to purify CD34+ cells, according to the manufacturer's protocol (Miltenyi Biotec, Auburn, CA, USA). .. All marrow samples were characterized in regard to clonal cytogenetic abnormalities using metaphase G banding, fluorescent in situ hybridization (FISH) or both in the clinical laboratory of the Seattle Cancer Care Alliance/FHCRC.

    FACS:

    Article Title: Effect of intravenous coadministration of human stroma cell lines on engraftment of long-term repopulating clonal myelodysplastic syndrome cells in immunodeficient mice
    Article Snippet: .. Bone marrow mononuclear cells and PB cells were separated by Ficoll–Hypaque gradient centrifugation and suspended in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum until use, or were subjected to magnetic-activated cell sorting to purify CD34+ cells, according to the manufacturer's protocol (Miltenyi Biotec, Auburn, CA, USA). .. All marrow samples were characterized in regard to clonal cytogenetic abnormalities using metaphase G banding, fluorescent in situ hybridization (FISH) or both in the clinical laboratory of the Seattle Cancer Care Alliance/FHCRC.

    Article Title: A Virally Encoded DeSUMOylase Activity Is Required for Cytomegalovirus Reactivation from Latency
    Article Snippet: Two washing steps in 2× saline sodium citrate buffer (5 min, 60°C) were followed by a PBS wash at room temperature and then staining as before to detect PML localization. .. Primary CD34+ hematopoietic cells were isolated from apheresis blood packs harvested from cranulocyte-colony-stimulating factor-mobilized donors using CD34+ magnetic activated cell sorting (MACS) separation (Miltenyi Biotec). .. Alternatively, CD34+ cells isolated from granulocyte-colony-stimulating factor (G-CSF)-mobilized healthy donors were purchased for use in some experiments (Lonza).The inhibition of isopeptidase activity was achieved using G5 (1 μM unless stated otherwise; Calbiochem) by addition of the chemical directly to the culture media 8 hr post-transfection or 3 hr prior to the addition of LPS in reactivation studies.

    Centrifugation:

    Article Title: Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform
    Article Snippet: Procedures for the use of UCB for this study were reviewed and approved by the medical ethics committee of Hannover Medical School. .. CB mononuclear cells were isolated using Ficoll density centrifugation and CD34 selection was performed using a CD34 microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. .. Purity of CD34+ cells was higher than 95% as determined by postenrichment flow cytometric analysis.

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    Miltenyi Biotec anti cd34 antibodies
    Endogenous GATA-1 expression is inversely related to CCR5 expression in human <t>CD34</t> + hematopoietic stem cells during DC differentiation. Purified and expanded primary human HSCs were cultured in stem cell media or DC-differentiating media and 2 endpoints
    Anti Cd34 Antibodies, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec cd34
    Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) <t>CD34</t> + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).
    Cd34, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The monoclonal antibody clone AC136 detects a class III epitope of the CD34 antigen This epitope is different than the one recognized by the clone used in the CD34 MicroBead
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    Endogenous GATA-1 expression is inversely related to CCR5 expression in human CD34 + hematopoietic stem cells during DC differentiation. Purified and expanded primary human HSCs were cultured in stem cell media or DC-differentiating media and 2 endpoints

    Journal:

    Article Title: Transcription factor GATA-1 potently represses the expression of the HIV-1 coreceptor CCR5 in human T cells and dendritic cells

    doi: 10.1182/blood-2005-03-0857

    Figure Lengend Snippet: Endogenous GATA-1 expression is inversely related to CCR5 expression in human CD34 + hematopoietic stem cells during DC differentiation. Purified and expanded primary human HSCs were cultured in stem cell media or DC-differentiating media and 2 endpoints

    Article Snippet: Human CD34+ HSCs were isolated from Ficoll-separated umbilical cord blood mononuclear cells by magnetic sorting using anti-CD34 antibodies conjugated to magnetic-activated cell sorting (MACS) beads (Miltenyi Biotech, Auburn, CA).

    Techniques: Expressing, Purification, Cell Culture

    Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).

    Journal: The Journal of Experimental Medicine

    Article Title: Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells

    doi:

    Figure Lengend Snippet: Chemotaxis of leukocytes in response to synthetic MIP-3α. ( a ) T cells; ( b ) monocytes; ( c ) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils ( closed circles ). ( d ) CD34 + DCs ( closed circles ) and peripheral blood monocyte–derived DCs ( open circles ). ( e ) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants ( open circles ); MIP-3α clone 16 transfectants ( closed circles ); and mock transfectants ( open squares ).

    Article Snippet: RT-PCR on RNA extracted from distinct human leukocyte populations indicated that DCCR2 mRNA expression was restricted to T cells, CD34+ DCs, and lung DCs but not on peripheral blood monocyte–derived DCs (Fig. ).

    Techniques: Chemotaxis Assay, Derivative Assay

    RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.

    Journal: The Journal of Experimental Medicine

    Article Title: Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3? from Lung Dendritic Cells

    doi:

    Figure Lengend Snippet: RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1 , lung DCs; lane 2 , peripheral blood monocyte–derived DCs; lane 3 , CD34 + DCs; lane 4 , CD4 T cells; lane 5 , CD8 T cells.

    Article Snippet: RT-PCR on RNA extracted from distinct human leukocyte populations indicated that DCCR2 mRNA expression was restricted to T cells, CD34+ DCs, and lung DCs but not on peripheral blood monocyte–derived DCs (Fig. ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Molecular Weight, Derivative Assay

    Mutation of the Putative Catalytic Site of LUNA Abrogates HCMV Reactivation from Latency (A) HFFs were infected at an MOI of 0.1 with WT Merlin, LUNA catalytic dead mutant (LUNA FUN-MUT ), or WT virus and the growth measured over 14 days. Supernatants were titered for infectious virus production every 2 days. (B) Western blotting on mock (M), LUNA SHORT (KO), WT, revertant (Rev), or LUNA functional mutant (FM) at 24 and 72 hpi for viral protein expression. (C and D) CD34 + cells latently infected with WT or a catalytic dead mutant of LUNA (LUNA FUN-MUT ) were differentiated to mature DCs. Induction of IE gene expression (C) or reactivation of virus by infectious center formation in co-cultures (D) was quantified by qRT-PCR or immuno-staining, respectively. Data are mean ± SD and represent triplicate analyses performed in three independent experiments. ∗∗ p

    Journal: Cell Reports

    Article Title: A Virally Encoded DeSUMOylase Activity Is Required for Cytomegalovirus Reactivation from Latency

    doi: 10.1016/j.celrep.2018.06.048

    Figure Lengend Snippet: Mutation of the Putative Catalytic Site of LUNA Abrogates HCMV Reactivation from Latency (A) HFFs were infected at an MOI of 0.1 with WT Merlin, LUNA catalytic dead mutant (LUNA FUN-MUT ), or WT virus and the growth measured over 14 days. Supernatants were titered for infectious virus production every 2 days. (B) Western blotting on mock (M), LUNA SHORT (KO), WT, revertant (Rev), or LUNA functional mutant (FM) at 24 and 72 hpi for viral protein expression. (C and D) CD34 + cells latently infected with WT or a catalytic dead mutant of LUNA (LUNA FUN-MUT ) were differentiated to mature DCs. Induction of IE gene expression (C) or reactivation of virus by infectious center formation in co-cultures (D) was quantified by qRT-PCR or immuno-staining, respectively. Data are mean ± SD and represent triplicate analyses performed in three independent experiments. ∗∗ p

    Article Snippet: Primary CD34+ hematopoietic cells were isolated from apheresis blood packs harvested from cranulocyte-colony-stimulating factor-mobilized donors using CD34+ magnetic activated cell sorting (MACS) separation (Miltenyi Biotec).

    Techniques: Mutagenesis, Infection, Western Blot, Functional Assay, Expressing, Quantitative RT-PCR, Immunostaining

    LUNA Is Required for Efficient Reactivation in CD34 + -Derived DCs (A) HFFs were infected at an MOI of 0.1 with Merlin, LUNA SHORT , or revertant virus, and the growth was measured over 14 days by titration of supernatants for infectious virus production every 2 days. (B) Viral gene expression was also assessed by western blotting of mock infected cells (M) or cells infected with LUNA SHORT (KO), wild-type (WT), or revertant (Rev) virus at 24 and 72 hrs post-infection (hpi). (C) CD34 + cells infected with WT, revertant, or LUNA SHORT were analyzed using qPCR for viral and cellular DNA levels at 3 dpi and then at 10 dpi after differentiation to an immature CD34 + -derived DC phenotype. (D) RNA isolated from WT revertant or LUNA SHORT infected CD34 + cells at 7 dpi were analyzed for UL138 gene expression using qRT-PCR. All samples were normalized to GAPDH and then expressed relative to WT virus. (E) CD34 + cells infected for 3 days to establish latency with WT, revertant, and LUNA SHORT viruses were differentiated and matured into CD34 + -derived DCs to induce reactivation and then co-cultured with fibroblasts. At 15 days, co-cultures were harvested and assayed for infectious virus production (IE forming units) on fresh fibroblasts. In (A) and (C)–(E), data are mean ± SD and represent triplicate analyses performed in two independent experiments. ∗ p

    Journal: Cell Reports

    Article Title: A Virally Encoded DeSUMOylase Activity Is Required for Cytomegalovirus Reactivation from Latency

    doi: 10.1016/j.celrep.2018.06.048

    Figure Lengend Snippet: LUNA Is Required for Efficient Reactivation in CD34 + -Derived DCs (A) HFFs were infected at an MOI of 0.1 with Merlin, LUNA SHORT , or revertant virus, and the growth was measured over 14 days by titration of supernatants for infectious virus production every 2 days. (B) Viral gene expression was also assessed by western blotting of mock infected cells (M) or cells infected with LUNA SHORT (KO), wild-type (WT), or revertant (Rev) virus at 24 and 72 hrs post-infection (hpi). (C) CD34 + cells infected with WT, revertant, or LUNA SHORT were analyzed using qPCR for viral and cellular DNA levels at 3 dpi and then at 10 dpi after differentiation to an immature CD34 + -derived DC phenotype. (D) RNA isolated from WT revertant or LUNA SHORT infected CD34 + cells at 7 dpi were analyzed for UL138 gene expression using qRT-PCR. All samples were normalized to GAPDH and then expressed relative to WT virus. (E) CD34 + cells infected for 3 days to establish latency with WT, revertant, and LUNA SHORT viruses were differentiated and matured into CD34 + -derived DCs to induce reactivation and then co-cultured with fibroblasts. At 15 days, co-cultures were harvested and assayed for infectious virus production (IE forming units) on fresh fibroblasts. In (A) and (C)–(E), data are mean ± SD and represent triplicate analyses performed in two independent experiments. ∗ p

    Article Snippet: Primary CD34+ hematopoietic cells were isolated from apheresis blood packs harvested from cranulocyte-colony-stimulating factor-mobilized donors using CD34+ magnetic activated cell sorting (MACS) separation (Miltenyi Biotec).

    Techniques: Derivative Assay, Infection, Titration, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Isolation, Quantitative RT-PCR, Cell Culture

    LUNA Isopeptidase Activity Is Required for HCMV Reactivation (A) CD34 + cells isolated from a healthy seropositive donor were cultured to immature DCs (iLC; lane 1) and then incubated with LPS to promote full virus reactivation (mLC; lanes 2–8). Prior to the addition of LPS, cells were incubated with mock (2), G5 isopeptidase inhibitor (25–0.25 μM; 3–5), or DMSO solvent (6–8) for 2 hr. Reactivation was measured using RT-PCR for IE72 expression on RNA isolated from cells 24 hr post-addition of LPS. Water (−ve) and cDNA from infected fibroblasts (+ve) served as PCR controls (lanes 9 and 10). (B) HFFs were incubated with 1 μM G5 and infected with HCMV at an MOI of 1 (high MOI) or 0.1 (low MOI) and then analyzed by immunofluorescence (IF) at 8 hr post-infection for IE gene expression, and percentage infection was calculated. (C) A qRT-PCR analysis for IE gene expression was performed on low-MOI infected HFF cells as described in (B). (D) CD34 + cells latently infected with WT or the LUNA protein disruption virus (LUNA SHORT ) were differentiated to immature DCs and then either incubated with DMSO or G5 prior to stimulation with LPS to fully reactivate virus. Twenty-four hours post-infection RNA was isolated and analyzed using IE qRT-PCR and quantified against a standard curve. Data are mean ± SD and represent triplicate analyses performed in three independent experiments. ∗ p

    Journal: Cell Reports

    Article Title: A Virally Encoded DeSUMOylase Activity Is Required for Cytomegalovirus Reactivation from Latency

    doi: 10.1016/j.celrep.2018.06.048

    Figure Lengend Snippet: LUNA Isopeptidase Activity Is Required for HCMV Reactivation (A) CD34 + cells isolated from a healthy seropositive donor were cultured to immature DCs (iLC; lane 1) and then incubated with LPS to promote full virus reactivation (mLC; lanes 2–8). Prior to the addition of LPS, cells were incubated with mock (2), G5 isopeptidase inhibitor (25–0.25 μM; 3–5), or DMSO solvent (6–8) for 2 hr. Reactivation was measured using RT-PCR for IE72 expression on RNA isolated from cells 24 hr post-addition of LPS. Water (−ve) and cDNA from infected fibroblasts (+ve) served as PCR controls (lanes 9 and 10). (B) HFFs were incubated with 1 μM G5 and infected with HCMV at an MOI of 1 (high MOI) or 0.1 (low MOI) and then analyzed by immunofluorescence (IF) at 8 hr post-infection for IE gene expression, and percentage infection was calculated. (C) A qRT-PCR analysis for IE gene expression was performed on low-MOI infected HFF cells as described in (B). (D) CD34 + cells latently infected with WT or the LUNA protein disruption virus (LUNA SHORT ) were differentiated to immature DCs and then either incubated with DMSO or G5 prior to stimulation with LPS to fully reactivate virus. Twenty-four hours post-infection RNA was isolated and analyzed using IE qRT-PCR and quantified against a standard curve. Data are mean ± SD and represent triplicate analyses performed in three independent experiments. ∗ p

    Article Snippet: Primary CD34+ hematopoietic cells were isolated from apheresis blood packs harvested from cranulocyte-colony-stimulating factor-mobilized donors using CD34+ magnetic activated cell sorting (MACS) separation (Miltenyi Biotec).

    Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Polymerase Chain Reaction, Immunofluorescence, Quantitative RT-PCR

    Alpharetroviral vectors containing a myeloproliferative sarcoma virus (MPSV) promoter and the modified feline endogenous retrovirus envelope glycoprotein RD114/TR deliver enhanced gene transfer into cord blood (CB)-derived CD34+ hematopoietic stem cells

    Journal: Molecular Therapy

    Article Title: Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform

    doi: 10.1038/mt.2016.89

    Figure Lengend Snippet: Alpharetroviral vectors containing a myeloproliferative sarcoma virus (MPSV) promoter and the modified feline endogenous retrovirus envelope glycoprotein RD114/TR deliver enhanced gene transfer into cord blood (CB)-derived CD34+ hematopoietic stem cells

    Article Snippet: CB mononuclear cells were isolated using Ficoll density centrifugation and CD34 selection was performed using a CD34 microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions.

    Techniques: Modification, Derivative Assay

    Transduction procedure and transgene positivity of human cord blood (CB)-derived CD34+ cells do neither impact the proliferation nor the differentiation pattern towards precursor T cells (preTs) in vitro when using enhanced green fluorescent protein (EGFP)

    Journal: Molecular Therapy

    Article Title: Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform

    doi: 10.1038/mt.2016.89

    Figure Lengend Snippet: Transduction procedure and transgene positivity of human cord blood (CB)-derived CD34+ cells do neither impact the proliferation nor the differentiation pattern towards precursor T cells (preTs) in vitro when using enhanced green fluorescent protein (EGFP)

    Article Snippet: CB mononuclear cells were isolated using Ficoll density centrifugation and CD34 selection was performed using a CD34 microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions.

    Techniques: Transduction, Derivative Assay, In Vitro

    Human cord blood (CB)-derived CD34+ cells are differentiated into precursor T cells (preTs) in vitro . ( a ) Human CB samples were obtained from consenting mothers and CD34+ cells consecutively isolated via positive selection by magnetic-activated cell sorting

    Journal: Molecular Therapy

    Article Title: Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform

    doi: 10.1038/mt.2016.89

    Figure Lengend Snippet: Human cord blood (CB)-derived CD34+ cells are differentiated into precursor T cells (preTs) in vitro . ( a ) Human CB samples were obtained from consenting mothers and CD34+ cells consecutively isolated via positive selection by magnetic-activated cell sorting

    Article Snippet: CB mononuclear cells were isolated using Ficoll density centrifugation and CD34 selection was performed using a CD34 microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions.

    Techniques: Derivative Assay, In Vitro, Isolation, Selection, FACS