14-0199-82

antibody from Invitrogen Antibodies

Targeting: CD19

Provider product page for 14-0199-82

Immunocytochemistry

Immunohistochemistry

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Antibody data

Antibody Data
Antigen structure
References [45]
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Validations
Immunocytochemistry [2]
Flow cytometry [1]
Other assay [32]

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Product number: 14-0199-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD19 Monoclonal Antibody (HIB19), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The HIB19 monoclonal antibody reacts with human CD19, a 95 kDa transmembrane glycoprotein. CD19 is expressed by B cells during all stages of development excluding the terminally differentiated plasma cells. Follicular dendritic cells also express CD19. Together CD21, CD81, Leu13, MHC class II, and CD19 form a multimolecular complex that associates with BCR. Signaling through CD19 induces tyrosine phosphorylation, calcium flux and proliferation of B cells. The SJ25C1 antibody and the HIB19 monoclonal antibody recognize overlapping epitopes. Applications Reported: The HIB19 antibody has been reported for use in flow cytometric analysis, and immunohistochemical staining of frozen tissue sections. It has also been reported in in vitro functional studies. (Please use Functional Grade Purified HIB19, Product # 16-0199, in functional assays. Fluorochrome-conjugated HIB19 is recommended for use in flow cytometry). Applications Tested: This HIB19 antibody has been tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at less than or equal to 1 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human, Mouse
Host: Mouse
Isotype: IgG
Antibody clone number: HIB19
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: 4° C

CD19 protein structure - 14-0199-82 shown in red.

Supportive validation

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Experimental details: Immunofluorescence analysis of B-lymphocyte antigen CD19 was performed using 70% confluent log phase Ramos cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with CD19 Monoclonal Antibody (HIB19), eBioscience™ (Product # 14-0199-80, 14-0199-82) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing membrane localization. Panel e represents no expression of CD19 in Jurkat cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

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Experimental details: Knockout of CD19 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR807388_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Immunofluorescence analysis was performed on wild type Raji cells (panel a,d), Raji Cas9 control cells (panel b,e) and Raji CD19KO cells (panel c, f). Cells were fixed, permeabilized, and labeled with CD19 Monoclonal Antibody (HIB19), eBioscience™ (Product # 14-0199-82, 1:100 dilution), followed by Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A32723, 1:2000). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal (panel c,f) upon CRISPR mediated knockout (KO) confirms that antibody is specific to CD19 (green). The images were captured at 60X magnification.

Supportive validation

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Supportive validation

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Experimental details: Figure 1 Human PC-Ms Accumulate in the Gut together with ME-M B Cells and Include a Circulating Counterpart Expressing Gut-Homing Receptors (A) Flow cytometry (FCM) of IgM and IgA on CD19 + CD38 hi CD10 - PCs from human ileum and colon. (B) Frequency of PC-Ms among total PCs, assessed by FCM. (C) FCM of selected surface molecules on naive (N) B cells, PC-Ms, and switched PCs (PC-SW) from human ileum. Numbers indicate mean fluorescence intensity (MFI). (D) Immunofluorescence analysis (IFA) of IgM (green), IgA (red), and DNA (blue) in human ileum and mouse small intestine (SI) lamina propria (LP). Original magnification, 20x. Scale bars, 50 mum. (E) Number of PC-Ms (top), PC-As (center) per mm 2 of LP, and PC-M/PC-A ratio (bottom) from human or mouse SI assessed following tissue IFA. Data summarize six different tissue samples where at least four high-power microscopic fields were analyzed. (F and G) Representative FCM (F) and frequency (G) of beta7 + CCR9 + cells in human circulating PC-Ms, PC-As, and PC-G/Es. (H) Representative FCM showing IgM versus IgD staining on CD19 + CD38 - CD10 - B cells from different human tissues. (I) Frequency of ME-M B cells from tissues shown in (H). (J) FCM of IgD, CD24, CD27, and CD148 on naive, ME-M, and ME-SW B cells from ileum. Data show one representative result (A, F, H) of 12 (B), 8 (G), or 52 (I) experiments or are from one experiment of at least 3 with similar results (C, D, J). Results are presented as mean +- SEM; two-tailed unpaired

Supportive validation

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Experimental details: Figure 4 D10N impairs CD40L-induced p65, p38 and Erk phosphorylation in PBMC-derived B-cells. PBMCs were cultured with (blue curves) or without (red shadow) 100 ng/ml CD40L for 15 minutes, followed by phospho-flow cytometric analysis of pp65, pp38 and pErk within the CD19 + gate. ( a ) Representative histograms (from one of two experiments performed for each genotype) are presented for PBMC-derived B-cells from Act1 D10N izygous (left panels), heterozygous (middle panels), and homozygous (right panels) individuals. ( b ) Correlations between MFI values for pp65, pp38 and pErk in stimulated CD19 + B-cells for all tested individuals (n = 63), Data are expressed as fold-change increases. Pearson correlations between pp65 and pp38, pp65 and pErk, pErk and pp38 were performed. Each symbol represents two experiments averaged for one donor ( c ) Phosphorylation of p65, p38 and Erk in B-cells from izygous, heterozygous, and homozygous groups in response to CD40L were expressed as fold MFI. Each symbol represents one individual donor. Summary data represented by horizontal lines represent mean +- SEM. * P < 0.05, ** P < 0.01, as assessed by one-way analysis of variance followed by Dunnett''s post hoc test.

Supportive validation

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Experimental details: Figure 3 Phenotypic characterization of circulating B cell subsets in patients and HC. (A) Gated by CD19 + B cells, IgD + CD27 - NB, IgD + CD27 + USM B cells, IgD - CD27 + SM B cells, and IgD - CD27 - DN B cells were identified. The graphs are representative for HC and patients with different duration. Mean value of each B cell subset's percentage is shown in the quadrants. (B) Statistical graphs for comparison of CD19 + B cells' percentages between patient groups and HC. (C-E) Statistical graphs for distinct CD19 + B cells subsets between patients (SP, n = 48; SD, n = 23; LD, n = 25; FF, n = 33; LF, n = 15.) and HC (n = 25). Error bars represent mean+-SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS P >= 0.05.

Supportive validation

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Experimental details: Figure 4 Immune profiling in DENV-treated whole blood cells 24 h postincubation. Following DENV (MOI = 1) coculture in 100 mu l of WB ex vivo for 24 h, (a) representative flow cytometric analysis and gating of various cells obtained from five cases, performed by staining for specific cell surface markers (CD4, CD8, CD11c, CD14, CD16, CD19, CD25, CD56, CD62L, and HLA-DR), in the DENV-infected and mock groups showed (b) the changes in the expression of specific immune cell populations as noted. (c) The results are shown as a percentage of the mean +- SD obtained from five cases. * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the mock group. R: region; WBC: white blood cell; bri: bright.

Supportive validation

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Experimental details: Figure 4 Increased B cells in mild and moderate patients and reduced CD8 + cytotoxic T cells in mild and convalescent patients. A. The stained PBMCs were gated on lymphocyte population and examined for the CD19 and CD3 expression in HC, mild, moderate and convalescent (upper FACS panel). One exemplary dot plot is shown per study group. The bar diagram shows CD3 - CD19 + B cells. *P-value

Supportive validation

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Experimental details: 10.1371/journal.pone.0067430.g005 Figure 5 Production of EV71 VP-1-specific IgG and IgM antibodies in patients with increased frequency of CD19 + HLA-DR + cells. (A) IgG and IgM antibodies were rapidly increased and were markedly higher in plasma from severe patients (n = 13) compared with normal controls (n = 7). The differences between two time points of every individuals were determined by Wilcoxon's Sign Rank Test. * P

Supportive validation

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Experimental details: Figure 1 Differentiation of Human PSC INS Reporter Cells (A) A diagram showing control (left panel) and experimental (right panel) INS reporter cells. (B) Immunohistochemistry (IHC) staining with an anti-human-C-peptide antibody in beta-like cells differentiated from INS:tdT, INS:CD19, and parental PSCs (right three panels) and undifferentiated PSCs (left panel). Scale bar, 50 mum. (C) Representative flow cytometry results of PSCs (left panels) and beta-like cells (right panels) differentiated from INS:tdT (top panel) and INS:CD19 (bottom panel), showing the GFP signal on the vertical axis and the tdTomato fluorescence signal (top panel) or anti-CD19 signal on the horizontal axis (bottom panel). (D) Quantifications of the proportion of INS reporter-expressing cells at the end of differentiation (n = 3). (E) Quantification of the luciferase signal in undifferentiated ESCs and beta-like cells differentiated from the INS reporter cells (n = 4). (F) In vitro glucose-stimulated insulin secretion (GSIS) assays of PSC-differentiated beta-like cells. The stimulation index shows the ratio of the amounts of secreted human insulin released when beta-like cells were treated with 20 mM glucose compared with 2.5 mM glucose (n = 4). (G) Confocal micrographs with an anti-tdTomato antibody (red) and an anti-C-peptide antibody (green) of beta-like cells differentiated from INS:tdT PSCs. Scale bar, 10 mum. (H) Confocal micrographs with an anti-CD19 antibody (red) and an anti-C-peptide antibody

Supportive validation

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Experimental details: Characterization and uptake of SHED-Exos. A Surface markers of SHED cells were analyzed by flow cytometry (FACS) and were positive for mesenchymal markers (CD44, CD105 and CD90) and negative for endothelial markers (CD45, CD19 and CD14). B The morphology of exosomes (indicated by arrows) was observed using a transmission electron microscope (TEM). Scale bar = 100 nm. C Particle size distribution of SHED-Exos assessed by nanoparticle tracking analysis (NTA). D Expression of exosome-specific CD63 and TSG101 validated using western blotting. E Efficient uptake of PKH67-labeled exosomes by HUVECs was detected at 24 h. Scale bars = 100 mum

Supportive validation

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Experimental details: Fig. 1 BM erythroid progenitors in DBA patients show compromised growth. a Schematic illustrating the experimental workflow. Bone marrow (BM) erythroid progenitors from healthy individuals (acting as normal controls, NC) and DBA patients, who were subcategorized into untreated (UT), glucocorticoid (GC)-responsive (GCR) and GC-non-responsive (GCNR) groups, were sorted using flow cytometry and processed for single-cell RNA-seq (scRNA-seq) using modified STRT-seq, followed by data analyses and experimental validation. b Representative FACS plots showing the gating strategies used for isolation of BFU-E cells from NCs, which were immunophenotypically defined as CD45 + CD3 - CD4 - CD14 - CD19 - CD41 - CD235a - CD123 - CD36 - CD34 + . c The percentage of colonies generated from FACS-sorted cells (BFU-E: CD45 + CD3 - CD4 - CD14 - CD19 - CD41 - CD235a - CD123 - CD36 - CD34 + ) from cord blood (CB) and BM mononuclear cells. We seeded the sorted cells from the CB of three individuals and BM of one individual due to the limited healthy BM sample. Results are represented as mean +- SEM. d Micrographs of representative BFU-E colonies differentiated from sorted BFU-E cells of NC and DBA UT patients on day 14 of the colony forming unit assay. Scale bars, 100 mum. See also Supplementary Fig. S1 , Tables S1 , 2 .

Supportive validation

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Experimental details: Figure 5 lp36 contributes to the association of B. burgdorferi with specific populations of dendritic cells. Human PBMCs (4x10 6 ) were co-incubated with 4x10 7 GFP-tagged B31 (black bars), A3-M9 lp36- (white bars) or A3-M9 lp36-/lp36+ (cross-hatched bars) B. burgdorferi for 6 hours at 4degC or 37degC. The percentages of GFP + mDC1s (CD19 - CD3 - BDCA2 - BDCA1 + ) ( A ), pDCs (CD19 - CD3 - BDCA2 + BDCA1 - ) or ( B ) mDC2s (CD19 - CD3 - BDCA3 + BDCA2 - ) ( C ) were determined by multiparameter flow cytometry. Dot plots representing 500,000 collected events are provided to illustrate gating strategies (left). Column graphs represent the mean and standard deviation of three biological replicates (right). Statistical analysis was performed using a one-way ANOVA with a Tukey's post-test for multiple comparisons.

Supportive validation

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Experimental details: Figure 2 Protein delivery efficiency of dNP2 in primary mouse and human immune cells. ( a , b ) Mouse primary splenocytes were isolated from 6-week-old female C57BL/6 mice and the cells were incubated with 5 muM EGFP, TAT- and dNP2-EGFP for 2 h. Intracellular fluorescence was analysed by flow cytometry and the data are represented as dot plots or mean fluorescence intensity (MFI) of the cells. ( c , d ) Human PBMCs were isolated from healthy donor blood and the cells were incubated with 5 muM EGFP, TAT-, dNP2-EGFP for 2 h. The data were analysed as described above. ( e ) Total splenocytes were incubated with 1 muM EGFP, TAT-, and dNP2-EGFP for 2 h. Cells were gated using markers specific for CD4 T cells (CD4 + ), B cells (CD19 + ), macrophages (CD11c lo CD11b hi F480 + ) and DCs (CD11c hi MHCII hi ). The EGFP signal in each cell population was then analysed by flow cytometric analysis. The relative MFI value was normalization to PBS treated cells. The red line indicates relative MFI of PBS-treated cells. ( f ) Total PBMCs were incubated with 1 muM EGFP, TAT-, and dNP2-EGFP for 2 h. Cells were gated with markers specific for CD4 T cells (CD4 + ), B cells (CD19 + ), macrophages (CD11b + ) and DCs (CD11c + ) and the data were then analysed as described above. ( g ) Time-lapse images of mouse CD4 T cells incubated with 1 muM EGFP, TAT- and dNP2-EGFP were acquired for 2 h (Scale bar, 15 mum) and ( h ) the average fluorescence intensities of 10 cells from each sample were calculate

Supportive validation

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Experimental details: Figure 1 Characteristics of BM-MSCs. (a) Representative morphology of BM-MSCs. Scale bar = 500 mu m. (b) Representative flow cytometric characterization of cell surface markers expressed on BM-MSCs. Isotypic controls were represented by the gray filled histograms.

Supportive validation

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Experimental details: Figure 2 Characteristics of transfected BM-MSCs. (a) Representative morphology of transfected BM-MSCs in negative control group. (b) More than 90% of BM-MSCs expressed GFP in negative control group. (c) Representative morphology of transfected BM-MSCs in knock-down group. (d) More than 90% of BM-MSCs expressed GFP in knock-down group. (e) Representative flow cytometric characterization of cell surface markers expressed on transfected BM-MSCs. Isotypic controls were represented by black line. The red line represented the negative control group and the blue line represented the knock-down group. (f) The knock-down of NR2F2 was confirmed by western blot analysis.

Supportive validation

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Experimental details: Figure 1 Phenotypic alterations in clinical ALL samples following CD19 CAR. ( a , c , e , g ) Fever curves, CRP values and clinical response to CD19 CAR treatment. ( b , d , f , h ) Bar graph demonstrating percent of cells expressing cell surface markers by flow cytometry in the bone marrow, gated on leukaemic blasts, with representative flow cytometry histograms of CD19 and CD11b on the right (grey, control; blue, pre-CAR; red, post CAR). ( a , b ) Pre-CAR sample and day +30 post-CAR sample from a patient who did not experience CRS following CD19 CAR. ( c , d ) Pre-CAR and +180 days post-CAR samples from a patient with normal karyotype multiple relapsed ALL, who had a severe CRS followed by an MRD-negative complete response with CD19 CAR, with no CAR + cells persisting beyond 60 days. ( e , f ) Pre-CAR and day +30 post-CAR samples from a patient with normal karyotype ALL who experienced a mild CRS and CAR expansion, but had persistent disease. ( g , h ) Pre-CAR and post-CAR samples from an infant with MLL-rearranged ALL, treated with CD19-41BB-zeta CAR, who relapsed with myeloid blasts.

Supportive validation

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Experimental details: Figure 5 Knockout of Pax5 or Ebf1 in E2a:PBX pre-B-cell ALL results in a phenotype mirroring relapse under CAR pressure. ( a ) Representative structure of Cd19 , Pax5 and Ebf1 , exons marked in tabs. Guide-RNA design for each knockout shown, with the CAS9 PAM site underlined, and cleavage site marked with a red arrowhead. ( b ) Flow cytometry plots for murine leukaemia cell lines E2a:PBX (upper panel) and Emu-RET 309 (bottom panel) following CRISPR/CAS9 knockout of CD19 (green), PAX5 (red), EBF1 (blue) or mock (grey shadow).

Supportive validation

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Experimental details: Figure 1 Two-step culture of hESCs generates immunophenotypic HSPCs that engraft poorly (A) Culture and isolation strategy for differentiating H1 hESCs to HSPCs. (B) Representative FACS plots from 11 experiments staining for CD34, CD90, CD38, CD45 and CD43 on hESC-derived CD34 + cells isolated from 2 week EBs (EB), and after 2 week maturation culture on OP9-M2 (EB-OP9), compared to cells from second trimester foetal liver that were isolated directly (FL) or cultured on OP9-M2 (FL-OP9). (C) Representative FACS plots from 9 experiments staining for CD38, CD34, CD90 and human foetal HSC self-renewal marker GPI-80 on hESC- and FL-derived cells. (D) Human engraftment in NSG mice with hESC-derived and FL-derived CD34 + cells, before and after OP9-M2 co-culture (individual values and mean are shown, n=5 EB, n=4 EB-OP9 and FL, and n=3 FL-OP9 transplanted mice, statistical significance was calculated using the Wilcoxon Rank Sum test, see Supplementary Table 7 for statistics source data). (E) Representative FACS plots of showing human CD45 + fraction in the mouse BM 12 weeks post-transplantation. Multi-lineage engraftment is assessed by CD19 and CD3 (B-and T-lymphoid), and CD66 and CD13 (myeloid) stainings.

Supportive validation

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Experimental details: Figure 4 Medial HOXA genes govern the function and identity of human foetal HSPCs (A, B) Microarray analysis of HOXA gene expression in CD34 + CD38 -/lo CD90 + GPI-80 + cells and their progeny (Mean values are shown, left, n=3 samples, GEO database GSE54316, and right, n=3 samples (CD34+CD38-CD90+) or 2 (CD34+CD38-CD90- and CD34+CD38-) GSE34974. (C) Schematic showing the strategy for lentiviral shRNA knockdown of HOXA5 or HOXA7 in FL-HSPCs. (D) Knockdown is confirmed using q-RT-PCR 1 week post-infection (mean +/- SD shown from n=3 different FL samples). (E) Representative FACS plots 30 days after HOXA5 or HOXA7 knockdown. (F) Quantification of HSPC subsets in empty-vector (CTR) and shRNA infected cells (shHOXA5 or shHOXA7) after 5, 14 and 30 days in culture (mean and SEM, n=6 independent experiments per condition for day 14 and n=3 for day 5 and 30). Statistical significance was assessed using Wilcoxon Signed Rank test. (G) Schematic showing the transplantation strategy with HOXA5 or HOXA7 knockdown FL-HSPCs. (H) Representative FACS plots from mouse BM 10 weeks post-transplantation assessing human CD45 + cells and multi-lineage engraftment (CD19 and CD3 for B-and T-lymphoid, and CD66 and CD33 for myeloid). (I) Quantification of human engraftment (n=9 mice per condition from 3 independent experiments Individual values and mean are shown.) Statistical significance was assessed using the Wilcoxon Rank Sum test (J) RNA-sequencing of HOXA7 knockdown FL-HSPCs at day 5 post-infectio

Supportive validation

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Experimental details: Figure 5 Overexpression of medial HOXA genes enhances proliferative potential in FL-HSPCs but does not confer HSC properties to hESC-HSPCs (A) Schematic showing the strategy for constitutive lentiviral overexpression of HOXA5 or HOXA7 in FUGW vectors in FL-HSPCs. (B) Representative FACS plots of FUGW empty vector, HOXA5- or HOXA7- overexpressing FL-HSPCs. (C, D) Expansion of total FL cells (C) or HSPCs (D) transduced with HOXA5- or HOXA7- overexpression vectors or empty vector control (CTR), (mean and SEM values from n=3 independent experiments; statistical significance was assessed using the paired Student's t -test. (E) q-RT-PCR confirming overexpression in transduced HSPCs sorted 1 week post-infection (n=1 experiment with 2 pooled donors). (F) CFU-Cs from 2000 HSPCs sorted after day 10 of infection with vectors overexpressing HOXA5 or HOXA7 or FUGW empty vector control (mean and SD values shown from n=4 transductions from 2 independent experiments, p-values shown correspond to CTR vs. OE-HOXA7). (G) Schematic showing the strategy for lentiviral overexpression of HOXA5 and/or HOXA7 and/or HOXA9 in FUGW vectors in EB CD34 + cells. (H) Representative examples of FACS plots of EB CD34 + cells overexpressing HOXA5 or HOXA7 or a combination of HOXA5 , HOXA7 and HOXA9 . Un-transduced FL is shown as a control. (I) Quantification of CD34 + CD38 -/lo CD45 + haematopoietic cells from (H), mean from n=4 independent experiments for CTR and n=3 for HOXA5/7/9, HOXA5, and HOXA7 at da

Supportive validation

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Experimental details: Figure 2 CESCs Shared Features with BM-MSCs Regarding Morphology, Stem Cell Surface Markers, and Differentiation Ability (A) H&E staining of the tissue section. (B) Morphology of CESCs in agarose after seeding 6 weeks later. (C) Histologic section stained with Alcian blue of chondrified pellets in which CESCs formed in chondrogenic induction medium after 3 weeks. (D) Alizarin red staining of CESCs that underwent osteogenic induction for 3 weeks. (E) Immunophenotypic profile of stem cells in CESCs by flow cytometric analysis. The green lines indicate the fluorescence intensity of cells stained with the corresponding antibodies, and the red lines represent isotype-matched negative control cells. (F) Percentages of CESCs expressing different stem cell markers (n = 6 independent experiments). Data represent the mean +- SD.