Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: Binding of AFM11 and CD19/CD3 BiTEs to recombinant antigens and CD3+ cells. (A) Recombinant CD19-Fc was directly immobilized on the Sensor Chip (CM5). AFM11-His was injected (100 nM), and during the dissociation phase the second antigen (CD3) was injected. (B) Jurkat cells or (C) Raji cells were stained with the indicated concentrations of AFM11-His, CD19M13-sm/CD3LCHC21-ktay BiTE, and CD19HD37/CD3TR66 BiTE on ice. Cell-surface bound antibodies were detected by anti-His mAb followed by FITC-conjugated goat anti-mouse IgG. Mean fluorescence intensities determined by flow cytometry were modeled as a sigmoidal dose-response by non-linear regression to calculate KD.

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Binding Assay, Recombinant, Injection, Staining, Fluorescence, Flow Cytometry

Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: Summary of the SPR measurements for AFM11-His and the comparator molecules. SPR measurements for binding to recombinant huCD19-Fc and huCD3γϵ; CD19 HD37 /CD3 TR66 BiTE was measured employing the same protocol as described. 21 The tabulated dissociation constant (K D ), association rate constant (k a ), and dissociation rate constant (k d ) were calculated using the global fit and the 1:1 Binding model across 6 concentration curves (0, 12.5, 25, 50, 100 and 200 nM) for AFM11-His and both comparator molecules

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Binding Assay, Recombinant, Concentration Assay

Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: (See previous page). Comparison of cytotoxic properties of AFM11-His and the CD19HD37/CD3TR66 BiTE on cell lines. (A) EC50 and (B) specific lysis were determined in cytotoxicity assays with calcein-labeled CD19+ MEC-1 target cells and primary human T cells as effector cells at an E:T ratio of 25:1 with incubation times ranging between 30 min and 300 min. At each time-point the EC50 (calculated by non-linear regression of the data modeled as a sigmoidal curve) and specific lysis for AFM11-His and CD19HD37/CD3TR66 BiTE were plotted as representative experiments. (C) AFM11-His-mediated target cell lysis by CD4+ and CD8+ T cell subsets. 4 and 23 h FACS-based cytotoxicity assays for AFM11-His and CD19HD37/CD3TR66 BiTE were performed with either unfractionated human T cells or enriched human CD4+ or CD8+ T cells as effector cells, and CD19+ NALM-6 cells as target cells at an E:T ratio of 5:1. The plotted values represent the mean and the SD of duplicate measurements. (D, E) Serial killing mediated by AFM11 and CD19HD37/CD3TR66 BiTE was demonstrated by FACS-based cytotoxicity assays. NALM-6 target cells and enriched human T cells were used at the indicated E:T ratios in the presence of increasing concentrations of AFM11-His and CD19HD37/CD3TR66 BiTE. After 23 h incubation, EC50 (D) and percentage of target cell lysis at 10 pM (E) were calculated for each E:T ratio and plotted as representative experiments.

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Comparison, Lysis, Labeling, Incubation

Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: AFM11 does not facilitate activation of human T cells in the absence of CD19+ target cells. Dose-responsive induction of CD25 by AFM11-His (A) and CD69 (B) expression on human T cells was assayed in cultures of human PBMC, B cell-depleted PBMC, and enriched T cells after 48 h incubation. Unfractionated PBMC contained 3.8% CD19+ cells. B cell-depleted cultures possessed 0.6% CD19+ cells, and the enriched T cell cultures contained 0.1% CD19+ cells. The kinetics of CD25 (C) and CD69 (D) expression induced by AFM11-His (10 ng/mL) were determined in human PBMC cultures containing 3.5% CD19+ cells and in B cell-depleted PBMC cultures containing < 0.1% CD19+ cells. A through D present representative experiments. (E) No induction of T cell proliferation in the absence of target cells. Unfractionated PBMC, B cell-depleted PBMC, and enriched unstimulated human T cells were cultured in the presence of increasing concentrations of AFM11-His or OKT3 for 4 days, and then pulsed overnight with BrdU. Incorporation of BrdU was assayed by a BrdU ELISA. Control (ctrl.) cells were cultured in the absence of antibodies. Mean absorbance, and SD of triplicates, measured at 450 nm are plotted. The relative fractions of CD19+ cells were 8% in unfractionated human PBMC, 0.8% in B cell-depleted PBMC, and < 0.1% in enriched T cells.

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Activation Assay, Expressing, Incubation, Cell Culture, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: Cytokine release by AFM11-His is strictly dependent on the presence of CD19+ target cells. (A) Cytokine release in cultures of PBMC, B cell-depleted PBMC, and enriched human T cells. Unfractionated human PBMC, B cell-depleted PBMC, and enriched T cells were cultured in the presence of: i) 10 µg/mL AFM11-His or OKT3, ii) 100 µg/mL PHA, or iii) without antibodies (w/o). After 48 h incubation IL-2, IL-6, IL-10, TNF, and IFN-γ in the cell-free culture supernatant were quantified by multiplexing and the results of representative experiments were plotted. (B) TNF, (C) IL-2, (D) IL-6, and (E) IFN-γ levels in culture supernatants of PBMC from 7 individual donors. Cytokine levels were determined by Luminex upon stimulation with antibodies in solution. PBMC were either stimulated with AFM11 (open circles) or OKT3 (closed triangles) at the indicated concentrations. IL-2 and TNF levels were measured after 24 h, IL-6 and IFN-γ levels were measured after 48 h. Background (asterisks) was measured in cultures supplemented with vehicle (formulation buffer) corresponding to the volume of highest AFM11 concentration used. Horizontal bars indicate mean values across the 7 donors.

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Cell Culture, Incubation, Multiplexing, Luminex, Formulation, Concentration Assay

Journal: Malaria Journal

Article Title: High purity high yield tandem B and T helper cell isolation for qRT-PCR analysis suitable for basically equipped laboratories

doi: 10.1186/s12936-018-2547-3

Figure Lengend Snippet: List of fluorophore-conjugated antibodies

Article Snippet: The magnetic B and Th cell isolation from whole blood was carried out using the EasySepTM HLA Chimerism Whole Blood CD19 Positive Selection Kit (Stemcell Technologies) and the EasySepTM Human Whole Blood CD4 Positive Selection Kit (Stemcell Technologies) [ , ].

Techniques:

Journal: Malaria Journal

Article Title: High purity high yield tandem B and T helper cell isolation for qRT-PCR analysis suitable for basically equipped laboratories

doi: 10.1186/s12936-018-2547-3

Figure Lengend Snippet: Flow cytometry analysis of purity and efficiency of B and Th cell isolation. Shown is a representative analysis of three independent isolations. a Flow cytometry analysis of whole blood after erythrocyte lysis (= before B cell isolation). Upper left: gating of live lymphocytes. Lower left: gating of single cells. Upper right: total T cells (CD3+) versus B cells (CD19+). Lower left: total T cells (CD3+) versus Th cells (CD3+CD4+). b Flow cytometry analysis of isolated CD19+ B cells (= positive fraction after B cell isolation). Upper left: gating of live lymphocytes. Lower left: gating of single cells. Upper middle: total T cells (CD3+) versus B cells (CD19+). Lower middle: total T cells (CD3+) versus Th cells (CD3+CD4+). Upper right: histogram of CD19 positive cells. Lower right: histogram of CD4 positive cells. c Flow cytometry analysis of isolated CD4+ Th cells (= positive fraction after Th cell isolation). Upper left: gating of live lymphocytes. Lower left: gating of single cells. Upper middle: total T cells (CD3+) versus B cells (CD19+). Lower middle: total T cells (CD3+) versus Th cells (CD3+CD4+). Upper right: histogram of CD19 positive cells. Lower right: histogram of CD4 positive cells. d Flow cytometry analysis of the negative fraction after Th cell isolation. Upper left: gating of live lymphocytes. Lower left: gating of single cells. Upper middle: total T cells (CD3+) versus B cells (CD19+). Lower middle: total T cells (CD3+) versus Th cells (CD3+CD4+). Upper right: histogram of CD19 positive cells. Lower right: histogram of CD4 positive cells

Article Snippet: The magnetic B and Th cell isolation from whole blood was carried out using the EasySepTM HLA Chimerism Whole Blood CD19 Positive Selection Kit (Stemcell Technologies) and the EasySepTM Human Whole Blood CD4 Positive Selection Kit (Stemcell Technologies) [ , ].

Techniques: Flow Cytometry, Cell Isolation, Lysis, Isolation

Journal: STAR Protocols

Article Title: Isolation of human and mouse myeloid-derived suppressor cells for metabolic analysis

doi: 10.1016/j.xpro.2022.101389

Figure Lengend Snippet:

Article Snippet: EasySepTM HLA Chimerism Whole Blood CD33 Positive Selection Kit , STEMCELL Technologies , Catalog # 17885.

Techniques: Purification, Blocking Assay, Recombinant, Staining, Adhesive, Isolation, Selection, Software, Cell Culture

Journal: STAR Protocols

Article Title: Isolation of human and mouse myeloid-derived suppressor cells for metabolic analysis

doi: 10.1016/j.xpro.2022.101389

Figure Lengend Snippet:

Article Snippet: EasySepTM HLA Chimerism Whole Blood CD33 Positive Selection Kit , STEMCELL Technologies , Catalog # 17885.

Techniques:

Journal: Oncotarget

Article Title: Combination of betulinic acid and chidamide inhibits acute myeloid leukemia by suppression of the HIF1α pathway and generation of reactive oxygen species

doi: 10.18632/oncotarget.21889

Figure Lengend Snippet: (a) Different AML cell lines and primary CD34+ were treated by either control (CTL) or BA (5μg/ml betulinic acid) for 24 hours and the mRNA for AHR was measured, n=4. * , P <0.05, vs CTL group; #, P <0.05, vs CTL in CD34+ group. (b) The THP1 cells were transfected with indicated AHR reporter constructs, then treated by either CTL or 15μg/ml BA for 24 hours, the AHR reporter activity was measured, n=5. * , P <0.05, vs AHR-2000 group; ¶, P <0.05, vs AHR-200 group; #, P <0.05, vs AHR-100 group. (c-j) The CD34+ or THP1 cells were treated by either BA or AZA (3μM) for 24 hours, and the cells were harvested for further analysis. (c) Representative picture for DNA methylation on AHR promoter. (d) Quantitation of (c), n=5. (e) ChIP analysis on AHR promoter, n=4. (f) Representative picture for AHR proteins. (g) Quantitation of (f), n=5. (h) AHR mRNA by qPCR, n=4. (i) AHR reporter activity, n=5. (j) ChIP analysis by Sp1 antibody on AHR promoter, n=4. * , P <0.05, vs CD34+ group; ¶, P <0.05, vs THP1 group. Results are expressed as mean ± SEM.

Article Snippet: The CD34 positive hematopoietic stem cells (CD34+ HSCs) were isolated from fresh whole blood (obtained from healthy donors) using the EasySepTM Whole Blood CD34 Selection Kit (#18086, STEMCELL Technologies).

Techniques: Control, Transfection, Construct, Activity Assay, DNA Methylation Assay, Quantitation Assay

Journal: Oncotarget

Article Title: Combination of betulinic acid and chidamide inhibits acute myeloid leukemia by suppression of the HIF1α pathway and generation of reactive oxygen species

doi: 10.18632/oncotarget.21889

Figure Lengend Snippet: The CD34+ cells or THP1 cells were treated with either control (THP1), THP1/BA (5μg/ml), or BA treatment plus Sp1 knockdown (THP1/BA/siSp1) for 24 hours in hypoxic conditions, and then cells were harvested for further analysis. (a) Representative pictures for Western Blotting analysis from nuclear extracts. (b) Quantitation of (a), n=5. (c) Representative pictures for IP/WB analysis from nuclear extracts, 10% H3 as input control. (d) Quantitation of (c), n=5. (e) HIF1α transcriptional activity assay, n=5. (f) ChIP analysis on VEGF promoter, n=4. (g) VEGF luciferase reporter assay, n=5. (h) VEGF mRNA level by qPCR, n=4. * , P <0.05, vs CD34+ group; ¶, P <0.05, vs THP1 group. Results are expressed as mean ± SEM.

Article Snippet: The CD34 positive hematopoietic stem cells (CD34+ HSCs) were isolated from fresh whole blood (obtained from healthy donors) using the EasySepTM Whole Blood CD34 Selection Kit (#18086, STEMCELL Technologies).

Techniques: Control, Knockdown, Western Blot, Quantitation Assay, Activity Assay, Luciferase, Reporter Assay

Journal: Oncotarget

Article Title: Combination of betulinic acid and chidamide inhibits acute myeloid leukemia by suppression of the HIF1α pathway and generation of reactive oxygen species

doi: 10.18632/oncotarget.21889

Figure Lengend Snippet: The CD34+ or THP1 cells were treated with either control, 15μg/ml BA alone (BA), 3μM CDM alone (CDM), combination of BA and CDM (BA/CDM), or BA/CDM combination in SOD2 overexpression cells (THP1/BA/CDM/SOD2) for 24 hours in hypoxic conditions, and the cells were harvested for further analysis. (a) SOD2 mRNA level by qPCR, n=4. (b) ROS formation, n=5. (c) 3-Nitrotyrosine formation, n=5. (d) 8-OHdG formation, n=5. (e) Representative pictures for γH2AX formation. (f) Quantitation of (e), n=5. (g) Apoptosis rate by TUNEL assay, n=5. (h) Caspase-3 activity, n=5. (i) Intracellular ATP level, n=5. (j) ∆ᴪm by TMRE fluorescence, n=5. * , P <0.05, vs CD34+ group; ¶, P <0.05, vs THP1 group; #, P <0.05, vs THP1/BA group. Results are expressed as mean ± SEM.

Article Snippet: The CD34 positive hematopoietic stem cells (CD34+ HSCs) were isolated from fresh whole blood (obtained from healthy donors) using the EasySepTM Whole Blood CD34 Selection Kit (#18086, STEMCELL Technologies).

Techniques: Control, Over Expression, Quantitation Assay, TUNEL Assay, Activity Assay, Fluorescence

Journal: Oncotarget

Article Title: Combination of betulinic acid and chidamide inhibits acute myeloid leukemia by suppression of the HIF1α pathway and generation of reactive oxygen species

doi: 10.18632/oncotarget.21889

Figure Lengend Snippet: The CD34+ or THP1 cells were treated with either control, 15μg/ml BA alone (BA), 3μM CDM alone (CDM), BA/CDM combination, or BA/CDM combination in HIF1C overexpression cells (THP1/BA/CDM/HIF1C) for 24 hours in hypoxic conditions, and the cells were harvested for further analysis. (a-b) Specific primers were designed to measure mRNA levels for endogenous HIF1α (a) and constitutive HIF1α (HIF1C) (b), n=4. (c) HIF1α transcriptional activity assay, n=5. (d) VEGF reporter activity, n=5. (e) VEGF mRNA level by qPCR, n=4. * , P <0.05, vs CD34+ group; ¶, P <0.05, vs THP1 group; #, P <0.05, vs THP1/BA group. Results are expressed as mean ± SEM.

Article Snippet: The CD34 positive hematopoietic stem cells (CD34+ HSCs) were isolated from fresh whole blood (obtained from healthy donors) using the EasySepTM Whole Blood CD34 Selection Kit (#18086, STEMCELL Technologies).

Techniques: Control, Over Expression, Activity Assay

Journal: Oncotarget

Article Title: Combination of betulinic acid and chidamide inhibits acute myeloid leukemia by suppression of the HIF1α pathway and generation of reactive oxygen species

doi: 10.18632/oncotarget.21889

Figure Lengend Snippet: The CD34+ or THP1 cells were treated with either control, 15μg/ml BA alone (BA), 3μM CDM alone (CDM), BA/CDM combination, or BA/CDM combination in SOD2 and HIF1C overexpression cells (THP1/BA/CDM/SOD2/HIF1C) for 24 hours in hypoxic conditions, and the cells were harvested for further analysis. (a-c) mRNA levels for SOD2 (a), endogenous HIF1α (b) and constitutive HIF1α (HIF1C) (c), n=4. (d) The cell proliferation analysis by thymidine incorporation, n=5. (e) Cell viability analysis by MTT assay, n=5. (f) Colony formation assay in soft agar, n=5. (g) Representative picture for colony formation (f). * , P <0.05, vs CD34+ group; ¶, P <0.05, vs THP1 group; #, P <0.05, vs THP1/BA group. Results are expressed as mean ± SEM.

Article Snippet: The CD34 positive hematopoietic stem cells (CD34+ HSCs) were isolated from fresh whole blood (obtained from healthy donors) using the EasySepTM Whole Blood CD34 Selection Kit (#18086, STEMCELL Technologies).

Techniques: Control, Over Expression, MTT Assay, Colony Assay