Structured Review

Becton Dickinson apc conjugated anti cd34
FACS and Fluidigm-based analysis of steady-state terminal granulopoiesis. a, FACS plots demonstrating the gating strategy used for sorting. The populations sorted for scRNA-Seq are indicated in shades of grey with accompanying cytospins. b, Violin plots of the gene and read-level metrics for each of the indicated libraries. Dashed lines indicate mean, lower, and upper quartiles. Sample size (n = # cells) displayed (top). c, Heatmap of gene expression defined by ICGS (Fluidigm C1, excluding cell-cycle genes) in scRNA-Seq data ( n = 516 cells). Each column represents a single cell and each row represents a single gene. ICGS clusters are annotated (top). HSCP-1, haematopoietic stem cell progenitor; NK, natural killer T-cell progenitor; HSCP-2; Meg, megakaryocytic; Eryth, erythrocytic; DC, dentritic cell; MDP, monocyte dendritic cell progenitor; Multi-Lin*, multi-lineage primed; cMoP, common monocyte progenitor; Mono, monocytic; MP, monocyte-committed progenitors; proNeu-1, neutrophil progenitor, proNeu-2; preNeu-1, neutrophil precursor, preNeu-2, preNeu-3; immNeu, immature neutrophil. d, Joint UMAP plot of scRNA-Seq data from c separated according to archival ( 29 ) and new Fludigm captures. e, Bar chart of the heatmap in c displaying the incidence and amplitude of selected genes. f, Heatmap of correlation between gene expression and each displayed cluster as generated by MarkerFinder (AltAnalyze software). g, FACS plots comparing expression of Ly6g with the indicated surface marker. FMO; fluorescence minus one control. h, Heatmap of cell cycle gene expression in ICGS-defined clusters in scRNA-Seq data ( n = 509 cells). Each column represents a single cell and each row represents a single gene. Gene expression clusters were generated in AltAnalyze and the ICGS clusters are annotated (top). FACS gates are annotated (bottom). LSK, Lineage Neg c-Kit + Sca-1 + ; CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; LK <t>CD34</t> + , Lineage Neg c-Kit + Sca-1 Neg CD34 + ; GMP-P CD11b + , granulocyte monocyte progenitor and precursor, GMP-P CD11b + Ly6g low , GMP-P CD11b + Ly6g high . Key genes are indicated (right). i, Scatter plot representation of scRNA-Seq data from h comparing the gene expression of G1 to S phase transition genes with G2 to M phase transition genes in each cell. Each point represents a single cell. j, Bar chart of the heatmap in h , displaying the incidence and amplitude of selected genes. k, Heatmap of correlation between gene expression and each displayed cluster as generated by MarkerFinder. l, Heatmap of enrichment for Gene Ontology biological processes enriched in the granulocytic clusters from the Fluidigm scRNA-Seq data from c with key processes indicated (right). m, Scatter plot representation of scRNA-Seq data from c where each point represents a single cell. Reads per cell indicate RSEM transcript aligned read counts for each cell library. Genes expressed per cell indicate the number of genes with a TPM > 1 for each single-cell library. Data in a and g are representative of three biological replicates. Data in f and k display Pearson correlation values.
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1) Product Images from "Mouse models of neutropenia reveal progenitor-stage-specific defects"

Article Title: Mouse models of neutropenia reveal progenitor-stage-specific defects

Journal: Nature

doi: 10.1038/s41586-020-2227-7

FACS and Fluidigm-based analysis of steady-state terminal granulopoiesis. a, FACS plots demonstrating the gating strategy used for sorting. The populations sorted for scRNA-Seq are indicated in shades of grey with accompanying cytospins. b, Violin plots of the gene and read-level metrics for each of the indicated libraries. Dashed lines indicate mean, lower, and upper quartiles. Sample size (n = # cells) displayed (top). c, Heatmap of gene expression defined by ICGS (Fluidigm C1, excluding cell-cycle genes) in scRNA-Seq data ( n = 516 cells). Each column represents a single cell and each row represents a single gene. ICGS clusters are annotated (top). HSCP-1, haematopoietic stem cell progenitor; NK, natural killer T-cell progenitor; HSCP-2; Meg, megakaryocytic; Eryth, erythrocytic; DC, dentritic cell; MDP, monocyte dendritic cell progenitor; Multi-Lin*, multi-lineage primed; cMoP, common monocyte progenitor; Mono, monocytic; MP, monocyte-committed progenitors; proNeu-1, neutrophil progenitor, proNeu-2; preNeu-1, neutrophil precursor, preNeu-2, preNeu-3; immNeu, immature neutrophil. d, Joint UMAP plot of scRNA-Seq data from c separated according to archival ( 29 ) and new Fludigm captures. e, Bar chart of the heatmap in c displaying the incidence and amplitude of selected genes. f, Heatmap of correlation between gene expression and each displayed cluster as generated by MarkerFinder (AltAnalyze software). g, FACS plots comparing expression of Ly6g with the indicated surface marker. FMO; fluorescence minus one control. h, Heatmap of cell cycle gene expression in ICGS-defined clusters in scRNA-Seq data ( n = 509 cells). Each column represents a single cell and each row represents a single gene. Gene expression clusters were generated in AltAnalyze and the ICGS clusters are annotated (top). FACS gates are annotated (bottom). LSK, Lineage Neg c-Kit + Sca-1 + ; CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; LK CD34 + , Lineage Neg c-Kit + Sca-1 Neg CD34 + ; GMP-P CD11b + , granulocyte monocyte progenitor and precursor, GMP-P CD11b + Ly6g low , GMP-P CD11b + Ly6g high . Key genes are indicated (right). i, Scatter plot representation of scRNA-Seq data from h comparing the gene expression of G1 to S phase transition genes with G2 to M phase transition genes in each cell. Each point represents a single cell. j, Bar chart of the heatmap in h , displaying the incidence and amplitude of selected genes. k, Heatmap of correlation between gene expression and each displayed cluster as generated by MarkerFinder. l, Heatmap of enrichment for Gene Ontology biological processes enriched in the granulocytic clusters from the Fluidigm scRNA-Seq data from c with key processes indicated (right). m, Scatter plot representation of scRNA-Seq data from c where each point represents a single cell. Reads per cell indicate RSEM transcript aligned read counts for each cell library. Genes expressed per cell indicate the number of genes with a TPM > 1 for each single-cell library. Data in a and g are representative of three biological replicates. Data in f and k display Pearson correlation values.
Figure Legend Snippet: FACS and Fluidigm-based analysis of steady-state terminal granulopoiesis. a, FACS plots demonstrating the gating strategy used for sorting. The populations sorted for scRNA-Seq are indicated in shades of grey with accompanying cytospins. b, Violin plots of the gene and read-level metrics for each of the indicated libraries. Dashed lines indicate mean, lower, and upper quartiles. Sample size (n = # cells) displayed (top). c, Heatmap of gene expression defined by ICGS (Fluidigm C1, excluding cell-cycle genes) in scRNA-Seq data ( n = 516 cells). Each column represents a single cell and each row represents a single gene. ICGS clusters are annotated (top). HSCP-1, haematopoietic stem cell progenitor; NK, natural killer T-cell progenitor; HSCP-2; Meg, megakaryocytic; Eryth, erythrocytic; DC, dentritic cell; MDP, monocyte dendritic cell progenitor; Multi-Lin*, multi-lineage primed; cMoP, common monocyte progenitor; Mono, monocytic; MP, monocyte-committed progenitors; proNeu-1, neutrophil progenitor, proNeu-2; preNeu-1, neutrophil precursor, preNeu-2, preNeu-3; immNeu, immature neutrophil. d, Joint UMAP plot of scRNA-Seq data from c separated according to archival ( 29 ) and new Fludigm captures. e, Bar chart of the heatmap in c displaying the incidence and amplitude of selected genes. f, Heatmap of correlation between gene expression and each displayed cluster as generated by MarkerFinder (AltAnalyze software). g, FACS plots comparing expression of Ly6g with the indicated surface marker. FMO; fluorescence minus one control. h, Heatmap of cell cycle gene expression in ICGS-defined clusters in scRNA-Seq data ( n = 509 cells). Each column represents a single cell and each row represents a single gene. Gene expression clusters were generated in AltAnalyze and the ICGS clusters are annotated (top). FACS gates are annotated (bottom). LSK, Lineage Neg c-Kit + Sca-1 + ; CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; LK CD34 + , Lineage Neg c-Kit + Sca-1 Neg CD34 + ; GMP-P CD11b + , granulocyte monocyte progenitor and precursor, GMP-P CD11b + Ly6g low , GMP-P CD11b + Ly6g high . Key genes are indicated (right). i, Scatter plot representation of scRNA-Seq data from h comparing the gene expression of G1 to S phase transition genes with G2 to M phase transition genes in each cell. Each point represents a single cell. j, Bar chart of the heatmap in h , displaying the incidence and amplitude of selected genes. k, Heatmap of correlation between gene expression and each displayed cluster as generated by MarkerFinder. l, Heatmap of enrichment for Gene Ontology biological processes enriched in the granulocytic clusters from the Fluidigm scRNA-Seq data from c with key processes indicated (right). m, Scatter plot representation of scRNA-Seq data from c where each point represents a single cell. Reads per cell indicate RSEM transcript aligned read counts for each cell library. Genes expressed per cell indicate the number of genes with a TPM > 1 for each single-cell library. Data in a and g are representative of three biological replicates. Data in f and k display Pearson correlation values.

Techniques Used: FACS, Expressing, Generated, Software, Marker, Fluorescence, Sublimation

Functional assessment of SCN patient-derived GFI1 variants and generation of Gfi1 ZnF-mutant mice. a, Representative FACS plots of lentiviral transduced LSK cells isolated from adult Irf8-eGFP transgenic mice with the %Irf8-eGFP high indicated. b, Graphical summary of FACS analysis of lentiviral transduced LSK cells isolated from adult Irf8-eGFP transgenic mice (top) with locations of the variants mapped to the GFI1 protein (bottom). EV; empty vector, *P2A; mutation not found in patients, italics; other variants detected in the same patient, bold; also found in patients diagnosed with a malignancy, ATG; start codon, TGA; stop codon, gray blocks; characterized protein domains, SNAG; Snail/Gfi1 family domain, SUMO; sumoylation domain, ZnF; zinc-finger. c, Schematic of the Gfi1 locus annotated with relevant features. Line with small arrows; intronic regions, numbered blocks; exons, black blocks; coding regions, gray blocks; noncoding regions, ATG; start codon, TGA; stop codon, SNAG; Snail/Gfi1 family domain-encoding region, ZFN; zinc-finger nucleases, large arrows; noncoding region used for genotyping. d-f, Schematic of the nucleotide changes made to the coding region and 3’ UTR to introduce the d, d’ N382S, e, e’ K403R, and f, f’ R412X mutations. g, Representative genotyping of the ZnF-mutant mice with or without restriction enzyme digestion. h, Representation of Sanger sequencing analysis of cDNA from adult whole bone marrow. Targeted wild-type nucleotides are underlined, mutated nucleotides are in bold, and red nucleotides indicate the location of the stop codon. i, Immunoblot analysis of two R412X founder lines using adult murine Lineage Neg bone marrow lysates. j, Graphical summary of FACS analysis of neonatal murine peripheral blood. k, Total cell counts per mL of neonatal peripheral blood as determined by FACS. l, Representative RNAscope images (left) and transcript quantitation (right) of the indicated transcripts in primary human CD34+ cells. The number of cells scored (left) and the number of donors tested (right) is indicated. m, Representative elecropherogram plots from Sanger sequencing of GFI1 RT-PCR products derived from iPSC. The arrow indicates the nucleotide substitution made to introduce the R412X mutation. n, Representative FACS plots of iPSC cells at the end of the 10-day hematopoietic differentiation protocol. o, Representative cytospin images of iPSC-derived neutrophils. Data in a, b, and l are representative of three biological replicates, data in j and k are representative of individual biological replicates, and is plotted as mean in a or as mean ± s.e.m in b, j, k, and l . * p
Figure Legend Snippet: Functional assessment of SCN patient-derived GFI1 variants and generation of Gfi1 ZnF-mutant mice. a, Representative FACS plots of lentiviral transduced LSK cells isolated from adult Irf8-eGFP transgenic mice with the %Irf8-eGFP high indicated. b, Graphical summary of FACS analysis of lentiviral transduced LSK cells isolated from adult Irf8-eGFP transgenic mice (top) with locations of the variants mapped to the GFI1 protein (bottom). EV; empty vector, *P2A; mutation not found in patients, italics; other variants detected in the same patient, bold; also found in patients diagnosed with a malignancy, ATG; start codon, TGA; stop codon, gray blocks; characterized protein domains, SNAG; Snail/Gfi1 family domain, SUMO; sumoylation domain, ZnF; zinc-finger. c, Schematic of the Gfi1 locus annotated with relevant features. Line with small arrows; intronic regions, numbered blocks; exons, black blocks; coding regions, gray blocks; noncoding regions, ATG; start codon, TGA; stop codon, SNAG; Snail/Gfi1 family domain-encoding region, ZFN; zinc-finger nucleases, large arrows; noncoding region used for genotyping. d-f, Schematic of the nucleotide changes made to the coding region and 3’ UTR to introduce the d, d’ N382S, e, e’ K403R, and f, f’ R412X mutations. g, Representative genotyping of the ZnF-mutant mice with or without restriction enzyme digestion. h, Representation of Sanger sequencing analysis of cDNA from adult whole bone marrow. Targeted wild-type nucleotides are underlined, mutated nucleotides are in bold, and red nucleotides indicate the location of the stop codon. i, Immunoblot analysis of two R412X founder lines using adult murine Lineage Neg bone marrow lysates. j, Graphical summary of FACS analysis of neonatal murine peripheral blood. k, Total cell counts per mL of neonatal peripheral blood as determined by FACS. l, Representative RNAscope images (left) and transcript quantitation (right) of the indicated transcripts in primary human CD34+ cells. The number of cells scored (left) and the number of donors tested (right) is indicated. m, Representative elecropherogram plots from Sanger sequencing of GFI1 RT-PCR products derived from iPSC. The arrow indicates the nucleotide substitution made to introduce the R412X mutation. n, Representative FACS plots of iPSC cells at the end of the 10-day hematopoietic differentiation protocol. o, Representative cytospin images of iPSC-derived neutrophils. Data in a, b, and l are representative of three biological replicates, data in j and k are representative of individual biological replicates, and is plotted as mean in a or as mean ± s.e.m in b, j, k, and l . * p

Techniques Used: Functional Assay, Derivative Assay, Mutagenesis, Mouse Assay, FACS, Isolation, Transgenic Assay, Plasmid Preparation, Zinc-Fingers, Introduce, Sequencing, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction

Cell states traversed during commitment to terminal granulopoiesis include a rare transitional state (bridging specification to commitment). a, FACS plot with annotated gates used to sort cells for scRNA-Seq (see Extended Data Fig. 4a ; granulocyte-monocyte-progenitor and precursor “GMP-P” gate). b, 3D FACS plot of GMP-P gate (see Extended Data Fig. 4a ). c, Quantified histological forms of FACS-sorted cells (from gates in a ). d, Schematic of fluorescent cell cycle reporter and representative FACS plots of GMP-P gate from Fucci2 transgenic mice. e, 3D FACS plots of bone marrow isolated from adult mice bearing Gfi1, Irf8 or Myc fluorescent-protein reporters, or Vcam1 expression. Events are pseudocolored for CD11b expression. f, Bar chart displaying incidence and amplitude for selected genes (Fluidigm C1; see Extended Data Fig. 4c )( n = 509 cells). ICGS clusters are annotated (top) FACS gates are annotated (bottom). g, Heatmap of correlation between gene expression and each displayed cluster (MarkerFinder). h, Bar chart displaying selected genes (same cells as in f ). i, Heatmap of correlation between gene expression and each displayed cluster (MarkerFinder). j-k, Plots of ADT UMIs (10X 3’: see Extended Data Fig. 5b , e ) where grey indicates all captured cells: j , red identifies cells within ICGS clusters (right). k, red or blue indicates the top 1% of cells expressing the indicated gene(s). l, URD pseudotime analysis (of data in Extended Data Fig. 5b ), and transcriptional features, or normalized Z-scores of enriched biological processes (of data in Extended Data Fig. 4c ). Data are representative of three independent biological replicates in a, b, d, and e. Data in c represents cumulative total cell numbers from one experiment using cells pooled from 3 male mice. Data in g and i display Pearson correlation values. LSK, Lineage Neg c-Kit + Sca-1 + ; CMP, common myeloid progenitor; LK CD34 + , Lineage Neg c-Kit + Sca-1 Neg CD34 + .
Figure Legend Snippet: Cell states traversed during commitment to terminal granulopoiesis include a rare transitional state (bridging specification to commitment). a, FACS plot with annotated gates used to sort cells for scRNA-Seq (see Extended Data Fig. 4a ; granulocyte-monocyte-progenitor and precursor “GMP-P” gate). b, 3D FACS plot of GMP-P gate (see Extended Data Fig. 4a ). c, Quantified histological forms of FACS-sorted cells (from gates in a ). d, Schematic of fluorescent cell cycle reporter and representative FACS plots of GMP-P gate from Fucci2 transgenic mice. e, 3D FACS plots of bone marrow isolated from adult mice bearing Gfi1, Irf8 or Myc fluorescent-protein reporters, or Vcam1 expression. Events are pseudocolored for CD11b expression. f, Bar chart displaying incidence and amplitude for selected genes (Fluidigm C1; see Extended Data Fig. 4c )( n = 509 cells). ICGS clusters are annotated (top) FACS gates are annotated (bottom). g, Heatmap of correlation between gene expression and each displayed cluster (MarkerFinder). h, Bar chart displaying selected genes (same cells as in f ). i, Heatmap of correlation between gene expression and each displayed cluster (MarkerFinder). j-k, Plots of ADT UMIs (10X 3’: see Extended Data Fig. 5b , e ) where grey indicates all captured cells: j , red identifies cells within ICGS clusters (right). k, red or blue indicates the top 1% of cells expressing the indicated gene(s). l, URD pseudotime analysis (of data in Extended Data Fig. 5b ), and transcriptional features, or normalized Z-scores of enriched biological processes (of data in Extended Data Fig. 4c ). Data are representative of three independent biological replicates in a, b, d, and e. Data in c represents cumulative total cell numbers from one experiment using cells pooled from 3 male mice. Data in g and i display Pearson correlation values. LSK, Lineage Neg c-Kit + Sca-1 + ; CMP, common myeloid progenitor; LK CD34 + , Lineage Neg c-Kit + Sca-1 Neg CD34 + .

Techniques Used: FACS, Transgenic Assay, Mouse Assay, Isolation, Expressing

Related Articles

Staining:

Article Title: CD150-dependent hematopoietic stem cells sensing of Brucella instructs myeloid commitment
Article Snippet: Flow CytometryFor FACS sorting and analysis we used a FACSAriaIII or a LSR-X20 (BD) and the FlowJo software v10 (Treestar). .. For HSC and progenitor analysis, total BM cells were depleted of mature cells using a direct lineage depletion kit (Miltenyi Biotec) and stained with antibodies anti-CD34-APC or anti-BV421 (BD Bioscience, cloneRAM34), anti-CD135-PE-CF594 or anti-PE (BD Bioscience, clone A2F10.1), anti-CD150-PE-Cy7 or anti-BV711 (BioLegend, clone TC15-12F12.2), anti-CD117-BV605 (BioLegend, clone 2B8), anti-Sca-1-PrcpCy5.5 or anti-PE (ThermoFischer Scientist, clone D7), anti-CD48-BV510 or anti-PE-Cy7 (BD Bioscience, clone HM48-1), anti-CD16/32-PE or anti-APC-Cy7 (BD Bioscience, clone 2.4G2). .. When needed, anti-CD45.1-APC or anti-BV421 (BD Bioscience, clone A20) and anti-CD45.2-FITC or anti-PrcpCy5.5 (BD Bioscience, clone 104) were added.

Article Title: Mouse models of neutropenia reveal progenitor-stage-specific defects
Article Snippet: After 5 days, culture medium was transitioned to RPMI 1640 medium supplemented with 10% FBS and 50 ng/ml G-CSF (Peprotech) and cultured for another 5 days. .. Cells were stained with PE-conjugated anti-CD45 (clone HI30, Becton, Dickenson, and Company), APC-conjugated anti-CD34 (clone 581, Becton, Dickenson, and Company), APC-Cy7-conjugated anti-CD11B (clone ICRF44, Becton, Dickenson, and Company), Vioblue-conjugated anti-CD14 (clone REA599, Miltenyi), and PE-Cy7-conjugated anti-CD16 (clone REA423, Miltenyi) prior to FACS analysis. .. To analyze GFI1 expression, cells were lysed in TriZol (Thermo Fisher) and total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).

Article Title: CD22-CAR T Cells Induce Remissions in CD19-CAR Naïve and Resistant B-ALL
Article Snippet: Specimens for flow cytometry were processed within 12 h of collection and stained with a panel of antibodies to quantitate leukemic burden and measure CAR T cells numbers. .. Briefly whole blood lysis was performed using ammonium chloride prior to staining for 30 minutes at room temperature with the following two cocktails (antibody concentration according to manufacturer’s recommendations): Cocktail A- CD16FITC (clone DJ130c, Dako), CD19PE (clone SJ25C1), CD3PerCP (clone SK7, BD), CD13PECy7 (L138, BD), CD34APC (clone 8G12, BD), CD14 APC H7 (MP9, BD), CD56v450 (clone B159, BD), CD45 v500 (clone HI30, BD); and Cocktail B-CD66bFITC (clone G10F5, BD), CD22PE (clone S-HCL-1, BD), CD34PerCP5.5 (clone8G12, BD), CD19PECy7 (clone SJ25C1, BD), CD24APC (clone SN3 A5-2H10, eBioscience), CD45 APC H7 (clone 2D1, BD), CD10BV421 (cloneHI10a, BD) and CD38BV510 (clone HB-7, Biolegend). .. At least 1 million cells were acquired per tube using an 8-color multiparametric approach on a 3-laser FACS Canto II (BD Biosciences, San Jose, CA) with DiVa 6.1.1 software and analyzed by FCS Express 4 software (DeNovo Software, Los Angeles, CA).

Article Title: Abcg2 Overexpression Represents a Novel Mechanism for Acquired Resistance to the Multi-Kinase Inhibitor Danusertib in BCR-ABL-Positive Cells In Vitro
Article Snippet: Cells cultured with 100 ng/mL colcemid (Invitrogen) were used to establish the CFSEmax quiescent cell population at all time points. .. CFSE+ cells of each condition were stained with an anti-CD34-APC-conjugated antibody and anti-active caspase-3-PE-conjugated antibody (BD PharMingen, San Diego) for quantification of apoptosis. ..

Labeling:

Article Title: TBP/TFIID-dependent activation of MyoD target genes in skeletal muscle cells
Article Snippet: The cell suspension was filtered through a 100 µm nylon cell strainer (BD Falcon, Tamaulipas, Mexico), followed by filtration through 40 µm nylon cell strainer (BD Falcon). .. Cells were labeled with primary antibodies: CD31-PacificBlue (RM5228, Life Technologies by Thermo Fisher Scientific, Waltham, MA, USA), CD45-eFluor450 (clone 30-F11, eBioscience, San Diego, CA, USA), Ter119-eFluor450 (clone TER-119, eBioscience), Sca-1-FITC (clone E13-161.7, BD Pharmingen, by BD Biosciences, San Josa, CA, USA), CD34-APC (clone RAM34, BD Pharmingen) and α7integrin-PE (clone R2F2, AbLab, Vancouver, Canada) for 30 min at 4°C, in a HBSS buffer with Mg2+ and Ca2+ containing 0.2% (w/v) BSA and 10 Units/ml Penicillin + 10 µg/ml Streptomycin. .. Cells were washed and resuspended in HBSS with Mg2+ and Ca2+ containing 0.2% (w/v) BSA and Penicillin /Streptomycin.

FACS:

Article Title: Mouse models of neutropenia reveal progenitor-stage-specific defects
Article Snippet: After 5 days, culture medium was transitioned to RPMI 1640 medium supplemented with 10% FBS and 50 ng/ml G-CSF (Peprotech) and cultured for another 5 days. .. Cells were stained with PE-conjugated anti-CD45 (clone HI30, Becton, Dickenson, and Company), APC-conjugated anti-CD34 (clone 581, Becton, Dickenson, and Company), APC-Cy7-conjugated anti-CD11B (clone ICRF44, Becton, Dickenson, and Company), Vioblue-conjugated anti-CD14 (clone REA599, Miltenyi), and PE-Cy7-conjugated anti-CD16 (clone REA423, Miltenyi) prior to FACS analysis. .. To analyze GFI1 expression, cells were lysed in TriZol (Thermo Fisher) and total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).

Lysis:

Article Title: CD22-CAR T Cells Induce Remissions in CD19-CAR Naïve and Resistant B-ALL
Article Snippet: Specimens for flow cytometry were processed within 12 h of collection and stained with a panel of antibodies to quantitate leukemic burden and measure CAR T cells numbers. .. Briefly whole blood lysis was performed using ammonium chloride prior to staining for 30 minutes at room temperature with the following two cocktails (antibody concentration according to manufacturer’s recommendations): Cocktail A- CD16FITC (clone DJ130c, Dako), CD19PE (clone SJ25C1), CD3PerCP (clone SK7, BD), CD13PECy7 (L138, BD), CD34APC (clone 8G12, BD), CD14 APC H7 (MP9, BD), CD56v450 (clone B159, BD), CD45 v500 (clone HI30, BD); and Cocktail B-CD66bFITC (clone G10F5, BD), CD22PE (clone S-HCL-1, BD), CD34PerCP5.5 (clone8G12, BD), CD19PECy7 (clone SJ25C1, BD), CD24APC (clone SN3 A5-2H10, eBioscience), CD45 APC H7 (clone 2D1, BD), CD10BV421 (cloneHI10a, BD) and CD38BV510 (clone HB-7, Biolegend). .. At least 1 million cells were acquired per tube using an 8-color multiparametric approach on a 3-laser FACS Canto II (BD Biosciences, San Jose, CA) with DiVa 6.1.1 software and analyzed by FCS Express 4 software (DeNovo Software, Los Angeles, CA).

Concentration Assay:

Article Title: CD22-CAR T Cells Induce Remissions in CD19-CAR Naïve and Resistant B-ALL
Article Snippet: Specimens for flow cytometry were processed within 12 h of collection and stained with a panel of antibodies to quantitate leukemic burden and measure CAR T cells numbers. .. Briefly whole blood lysis was performed using ammonium chloride prior to staining for 30 minutes at room temperature with the following two cocktails (antibody concentration according to manufacturer’s recommendations): Cocktail A- CD16FITC (clone DJ130c, Dako), CD19PE (clone SJ25C1), CD3PerCP (clone SK7, BD), CD13PECy7 (L138, BD), CD34APC (clone 8G12, BD), CD14 APC H7 (MP9, BD), CD56v450 (clone B159, BD), CD45 v500 (clone HI30, BD); and Cocktail B-CD66bFITC (clone G10F5, BD), CD22PE (clone S-HCL-1, BD), CD34PerCP5.5 (clone8G12, BD), CD19PECy7 (clone SJ25C1, BD), CD24APC (clone SN3 A5-2H10, eBioscience), CD45 APC H7 (clone 2D1, BD), CD10BV421 (cloneHI10a, BD) and CD38BV510 (clone HB-7, Biolegend). .. At least 1 million cells were acquired per tube using an 8-color multiparametric approach on a 3-laser FACS Canto II (BD Biosciences, San Jose, CA) with DiVa 6.1.1 software and analyzed by FCS Express 4 software (DeNovo Software, Los Angeles, CA).

Flow Cytometry:

Article Title: Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera
Article Snippet: .. Cells were analyzed by flow cytometry after the addition of 10 μl (5 μl of APC antibodies) of markers; CD71-FITC, CD45-PerCP, CD44-PE, annexin V-APC, CD41a-FITC, and CD34-APC (BD Biosciences Europe, Oxford, UK), incubated for 30 minutes at 4°C, and washed with PBS or binding buffer 1X (BD Biosciences Europe, Oxford, UK) (in the case of annexin) before analysis. .. Samples were analyzed using a flow cytometer FACSCalibur (Beckman Coulter, Fullerton, CA).

Incubation:

Article Title: Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera
Article Snippet: .. Cells were analyzed by flow cytometry after the addition of 10 μl (5 μl of APC antibodies) of markers; CD71-FITC, CD45-PerCP, CD44-PE, annexin V-APC, CD41a-FITC, and CD34-APC (BD Biosciences Europe, Oxford, UK), incubated for 30 minutes at 4°C, and washed with PBS or binding buffer 1X (BD Biosciences Europe, Oxford, UK) (in the case of annexin) before analysis. .. Samples were analyzed using a flow cytometer FACSCalibur (Beckman Coulter, Fullerton, CA).

Binding Assay:

Article Title: Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera
Article Snippet: .. Cells were analyzed by flow cytometry after the addition of 10 μl (5 μl of APC antibodies) of markers; CD71-FITC, CD45-PerCP, CD44-PE, annexin V-APC, CD41a-FITC, and CD34-APC (BD Biosciences Europe, Oxford, UK), incubated for 30 minutes at 4°C, and washed with PBS or binding buffer 1X (BD Biosciences Europe, Oxford, UK) (in the case of annexin) before analysis. .. Samples were analyzed using a flow cytometer FACSCalibur (Beckman Coulter, Fullerton, CA).

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    Becton Dickinson apc conjugated anti human cd34 mab
    Representative FACS profiles of CB-derived 18Lin − CD45 + <t>CD34</t> +/− CD133 +/− cells. ( a ) The FSC/SSC profile of immunomagnetically separated Lin − cells. The R1 gate was set on the blast–lymphocyte window. ( b ) The R2 gate was set on the 18Lin − living cells. ( c ) The 18Lin − living cells (R2) were subdivided into two fractions: 18Lin − CD45 + CD34 + (R3) and CD34 − (R4) cells, according to their expression levels of CD34. The definitions of CD34 +/− cells are as follows: the CD34 + fraction contains cells expressing maximum <t>APC</t> fluorescence intensity (FI) to 5% the level of FI. The CD34 − level of FI was determined based on the Fluorescence Minus One controls. ( d ) The 18Lin − CD34 + cells residing in the R3 gate were further subdivided into two fractions: 18Lin − CD45 + CD133 + (R5) and CD133 − (R6) cells, according to their expression levels of CD133. The definitions of CD133 +/− cells are as follows: the CD133 + fraction contains cells expressing maximum PE FI to 15% the level of FI and the CD133 − level of FI was determined based on the Fluorescence Minus One controls. ( e ) The R4-gated cells were further subdivided into two fractions: 18Lin − CD45 + CD34 − CD133 + (R7) and CD133 − (R8) cells. The definitions of CD133 +/− cells are the same as described above.
    Apc Conjugated Anti Human Cd34 Mab, supplied by Becton Dickinson, 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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    Becton Dickinson alexa fluor 647 conjugated anti mouse cd34
    DJ001 promotes human HSC regeneration following irradiation. a At left, representative flow cytometric analysis <t>of</t> CD34 + CD38 - cells at 36 h after 300 cGy irradiation of BM CD34 + cells and culture with media ± 1 µg/mL DJ001. At right, mean percentages and numbers of CD34 + CD38 − cells in each group (Day 0, n = 6; media, DJ001, n = 10). One-way ANOVA with Tukey’s multiple comparison test. b Numbers of CFU-GEMMs within cultures of CD34 + cells at 72 h after 300 cGy and culture with media ± DJ001 ( n = 6). c At left, representative flow cytometric analysis of Annexin V/7AAD staining of CD34 + cells at 24 h after 300 cGy and culture with media ± DJ001 ± EHT1864. At right, percentages of live and apoptotic CD34 + CD38 − cells in each condition (media and DJ001, n = 10; DJ001 + EHT1864, n = 5). One-way ANOVA with Tukey’s multiple comparison test. d Fold changes (2 −ΔΔCt ) in BCL2L1 and MCL-1 gene expression in CD34 + CD38 − cells at 12 h after 300 cGy in media ± DJ001 ( n = 3). Gene transcript changes were normalized to GAPDH and media treatment. Two-way ANOVA with Sidak’s multiple comparison test. e Schematic representation of NSG mice transplantation assay using the progeny of human BM CD34 + cells irradiated with 300 cGy and treated with or without DJ001 × 36 h. f Representative flow cytometric analysis of human CD45 + cells, human CD34 + cells, human CD19 + B cells, human CD33 + myeloid cells, and human CD3 + T cells engraftment in the BM of NSG mice at 12 weeks post transplantation. g Percent engraftment of human hematopoietic cell subsets in the BM of NSG mice at 12 weeks post transplantation ( n = 11–12/group). Source data are provided as Source Data File. * P
    Alexa Fluor 647 Conjugated Anti Mouse Cd34, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 647 conjugated anti mouse cd34/product/Becton Dickinson
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    Becton Dickinson apc mouse anti human cd34
    Mesenchymal stem cell (MSC) characterization in normoxic and hypoxic conditions. a Positive MSC markers (CD105, CD73, and CD90) and b negative MSC markers (CD,14, <t>CD34,</t> CD45)
    Apc Mouse Anti Human Cd34, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apc mouse anti human cd34/product/Becton Dickinson
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    Image Search Results


    Representative FACS profiles of CB-derived 18Lin − CD45 + CD34 +/− CD133 +/− cells. ( a ) The FSC/SSC profile of immunomagnetically separated Lin − cells. The R1 gate was set on the blast–lymphocyte window. ( b ) The R2 gate was set on the 18Lin − living cells. ( c ) The 18Lin − living cells (R2) were subdivided into two fractions: 18Lin − CD45 + CD34 + (R3) and CD34 − (R4) cells, according to their expression levels of CD34. The definitions of CD34 +/− cells are as follows: the CD34 + fraction contains cells expressing maximum APC fluorescence intensity (FI) to 5% the level of FI. The CD34 − level of FI was determined based on the Fluorescence Minus One controls. ( d ) The 18Lin − CD34 + cells residing in the R3 gate were further subdivided into two fractions: 18Lin − CD45 + CD133 + (R5) and CD133 − (R6) cells, according to their expression levels of CD133. The definitions of CD133 +/− cells are as follows: the CD133 + fraction contains cells expressing maximum PE FI to 15% the level of FI and the CD133 − level of FI was determined based on the Fluorescence Minus One controls. ( e ) The R4-gated cells were further subdivided into two fractions: 18Lin − CD45 + CD34 − CD133 + (R7) and CD133 − (R8) cells. The definitions of CD133 +/− cells are the same as described above.

    Journal: Leukemia

    Article Title: CD133 is a positive marker for a distinct class of primitive human cord blood-derived CD34-negative hematopoietic stem cells

    doi: 10.1038/leu.2013.326

    Figure Lengend Snippet: Representative FACS profiles of CB-derived 18Lin − CD45 + CD34 +/− CD133 +/− cells. ( a ) The FSC/SSC profile of immunomagnetically separated Lin − cells. The R1 gate was set on the blast–lymphocyte window. ( b ) The R2 gate was set on the 18Lin − living cells. ( c ) The 18Lin − living cells (R2) were subdivided into two fractions: 18Lin − CD45 + CD34 + (R3) and CD34 − (R4) cells, according to their expression levels of CD34. The definitions of CD34 +/− cells are as follows: the CD34 + fraction contains cells expressing maximum APC fluorescence intensity (FI) to 5% the level of FI. The CD34 − level of FI was determined based on the Fluorescence Minus One controls. ( d ) The 18Lin − CD34 + cells residing in the R3 gate were further subdivided into two fractions: 18Lin − CD45 + CD133 + (R5) and CD133 − (R6) cells, according to their expression levels of CD133. The definitions of CD133 +/− cells are as follows: the CD133 + fraction contains cells expressing maximum PE FI to 15% the level of FI and the CD133 − level of FI was determined based on the Fluorescence Minus One controls. ( e ) The R4-gated cells were further subdivided into two fractions: 18Lin − CD45 + CD34 − CD133 + (R7) and CD133 − (R8) cells. The definitions of CD133 +/− cells are the same as described above.

    Article Snippet: The cells were also stained with a PE-Cy7-conjugated anti-mouse CD45.1 mAb (Beckman Coulter); fluorescein isothiocyanate-conjugated anti-human CD19 (eBioscience), CD11b (Beckman Coulter) and CD235a (DAKO) mAbs; PE-conjugated anti-human CD33, CD14 and CD41 (Beckman Coulter); and an APC-conjugated anti-human CD34 mAb (BD Biosciences) for the detection of human stem/progenitor, B-lymphoid and myeloid hematopoietic cells.

    Techniques: FACS, Derivative Assay, Expressing, Fluorescence

    DJ001 promotes human HSC regeneration following irradiation. a At left, representative flow cytometric analysis of CD34 + CD38 - cells at 36 h after 300 cGy irradiation of BM CD34 + cells and culture with media ± 1 µg/mL DJ001. At right, mean percentages and numbers of CD34 + CD38 − cells in each group (Day 0, n = 6; media, DJ001, n = 10). One-way ANOVA with Tukey’s multiple comparison test. b Numbers of CFU-GEMMs within cultures of CD34 + cells at 72 h after 300 cGy and culture with media ± DJ001 ( n = 6). c At left, representative flow cytometric analysis of Annexin V/7AAD staining of CD34 + cells at 24 h after 300 cGy and culture with media ± DJ001 ± EHT1864. At right, percentages of live and apoptotic CD34 + CD38 − cells in each condition (media and DJ001, n = 10; DJ001 + EHT1864, n = 5). One-way ANOVA with Tukey’s multiple comparison test. d Fold changes (2 −ΔΔCt ) in BCL2L1 and MCL-1 gene expression in CD34 + CD38 − cells at 12 h after 300 cGy in media ± DJ001 ( n = 3). Gene transcript changes were normalized to GAPDH and media treatment. Two-way ANOVA with Sidak’s multiple comparison test. e Schematic representation of NSG mice transplantation assay using the progeny of human BM CD34 + cells irradiated with 300 cGy and treated with or without DJ001 × 36 h. f Representative flow cytometric analysis of human CD45 + cells, human CD34 + cells, human CD19 + B cells, human CD33 + myeloid cells, and human CD3 + T cells engraftment in the BM of NSG mice at 12 weeks post transplantation. g Percent engraftment of human hematopoietic cell subsets in the BM of NSG mice at 12 weeks post transplantation ( n = 11–12/group). Source data are provided as Source Data File. * P

    Journal: Nature Communications

    Article Title: PTPσ inhibitors promote hematopoietic stem cell regeneration

    doi: 10.1038/s41467-019-11490-5

    Figure Lengend Snippet: DJ001 promotes human HSC regeneration following irradiation. a At left, representative flow cytometric analysis of CD34 + CD38 - cells at 36 h after 300 cGy irradiation of BM CD34 + cells and culture with media ± 1 µg/mL DJ001. At right, mean percentages and numbers of CD34 + CD38 − cells in each group (Day 0, n = 6; media, DJ001, n = 10). One-way ANOVA with Tukey’s multiple comparison test. b Numbers of CFU-GEMMs within cultures of CD34 + cells at 72 h after 300 cGy and culture with media ± DJ001 ( n = 6). c At left, representative flow cytometric analysis of Annexin V/7AAD staining of CD34 + cells at 24 h after 300 cGy and culture with media ± DJ001 ± EHT1864. At right, percentages of live and apoptotic CD34 + CD38 − cells in each condition (media and DJ001, n = 10; DJ001 + EHT1864, n = 5). One-way ANOVA with Tukey’s multiple comparison test. d Fold changes (2 −ΔΔCt ) in BCL2L1 and MCL-1 gene expression in CD34 + CD38 − cells at 12 h after 300 cGy in media ± DJ001 ( n = 3). Gene transcript changes were normalized to GAPDH and media treatment. Two-way ANOVA with Sidak’s multiple comparison test. e Schematic representation of NSG mice transplantation assay using the progeny of human BM CD34 + cells irradiated with 300 cGy and treated with or without DJ001 × 36 h. f Representative flow cytometric analysis of human CD45 + cells, human CD34 + cells, human CD19 + B cells, human CD33 + myeloid cells, and human CD3 + T cells engraftment in the BM of NSG mice at 12 weeks post transplantation. g Percent engraftment of human hematopoietic cell subsets in the BM of NSG mice at 12 weeks post transplantation ( n = 11–12/group). Source data are provided as Source Data File. * P

    Article Snippet: For MEP, CMP, GMP, and CLP cell analysis, BM cells were stained with APC-Cy7-conjugated anti-mouse Sca1 (BD Biosciences, #560654, 1:100), PE-conjugated anti-mouse ckit (BD Biosciences, #553355, 1:100), V450 lineage cocktail (BD Biosciences, #561301; 1:10), Alexa Fluor 488-conjugated anti-mouse CD127 (BD Biosciences, #561533, 1:100), Alexa Fluor 647-conjugated anti-mouse CD34 (BD Biosciences, #560230; 1:100), and BV605-conjugated anti-mouse CD16/32 antibodies (BD Biosciences, #563006; 1:100).

    Techniques: Irradiation, Staining, Expressing, Mouse Assay, Transplantation Assay

    Mesenchymal stem cell (MSC) characterization in normoxic and hypoxic conditions. a Positive MSC markers (CD105, CD73, and CD90) and b negative MSC markers (CD,14, CD34, CD45)

    Journal: Stem Cell Research & Therapy

    Article Title: Hypoxic preconditioning induces epigenetic changes and modifies swine mesenchymal stem cell angiogenesis and senescence in experimental atherosclerotic renal artery stenosis

    doi: 10.1186/s13287-021-02310-z

    Figure Lengend Snippet: Mesenchymal stem cell (MSC) characterization in normoxic and hypoxic conditions. a Positive MSC markers (CD105, CD73, and CD90) and b negative MSC markers (CD,14, CD34, CD45)

    Article Snippet: Conversely, MSC were expected to not express CD45 (Abcam, Cat. # ab51482), CD34 (BD Biosciences, San Jose, CA, Cat. # 340441), or CD14 (Abcam, Cat. # ab82012).

    Techniques:

    Phenotypic diversity of acute myeloid leukemia (AML) leukemic stem cells (LSCs). (A) CD34 and CD38 surface marker profiles of all 52 patients. (B) Gating strategy for AML-LSCs. (C) Three distinct populations were analyzed as LSC phenotypes: left, multipotent progenitor (MPP)-like LSC; middle, lymphoid primed multipotent progenitor (LMPP)-like LSC; right, granulocyte-macrophage progenitors (GMP)-like LSC.

    Journal: The Korean Journal of Internal Medicine

    Article Title: Leukemic stem cell phenotype is associated with mutational profile in acute myeloid leukemia

    doi: 10.3904/kjim.2020.014

    Figure Lengend Snippet: Phenotypic diversity of acute myeloid leukemia (AML) leukemic stem cells (LSCs). (A) CD34 and CD38 surface marker profiles of all 52 patients. (B) Gating strategy for AML-LSCs. (C) Three distinct populations were analyzed as LSC phenotypes: left, multipotent progenitor (MPP)-like LSC; middle, lymphoid primed multipotent progenitor (LMPP)-like LSC; right, granulocyte-macrophage progenitors (GMP)-like LSC.

    Article Snippet: Cells were stained with following anti-human monoclonal antibodies: CD45-APC/cy7 (557833), CD34-APC (555824), CD38-BV421 (562445), CD90-PE (555596), CD123-PE/Cy7 (560826), and CD45RA-PerCP/Cy5.5 (563429) (BD Bioscience).

    Techniques: Marker