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AllCells LLC peripheral blood mononuclear cells pbmcs
Inhibition of GSK3 with CHIR99021 enhances NK cell cytokine production and anti-tumor function in vitro CD3/19-depleted <t>PBMCs</t> from <t>CMV</t> seropositive donors were cultured overnight with 10 ng/ml IL-15, for 7 days with 10 ng/ml IL-15 and DMSO or for 7 days with 10 ng/ml IL-15 and 5 μM CHIR99021 prior to functional analysis. A , NK cells cultured in the conditions above were incubated with K562 cells at the indicated effector:target ratios followed by the addition of Caspase-3/7 Green Detection Reagent. FACS was used to determine the percentage of K562 cells undergoing apoptosis ( n = 15). Results are from 3 independent experiments and are shown as mean ± SEM. Paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01. NK cells cultured in the conditions above were incubated with K562 cells at a 1:1 ratio, and intracellular FACS was used to measure the percentage of CD3 − CD56 + NK cells positive for B , TNF and C , IFN-γ (right panel) ( n = 7). Results are from 4 independent experiments and are shown as mean ± SEM. Paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01. NK cells ( n = 3) cultured in the conditions above were incubated with D , A549 cells, E , SKOV-3 cells and F , PANC-1 cells expressing NucLight Red fluorescent protein at a 3:1 ratio with the addition of a control anti-CD20 antibody. Parallel assays with NK cells ( n = 3) cultured in the conditions above were performed with G , A549 cells, H, SKOV-3 cells and I, PANC-1 cells expressing NucLight Red fluorescent protein at a 3:1 ratio with the addition of either anti-Her2 or anti-EGFR antibodies. An IncuCyte Imaging System was used to measure target cell killing over 48 hours. Results are from 3 independent experiments and are shown as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons tests was used to determine significance.
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1) Product Images from "GSK 3 inhibition drives maturation of NK cells and enhances their antitumor activity"

Article Title: GSK 3 inhibition drives maturation of NK cells and enhances their antitumor activity

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-17-0799

Inhibition of GSK3 with CHIR99021 enhances NK cell cytokine production and anti-tumor function in vitro CD3/19-depleted PBMCs from CMV seropositive donors were cultured overnight with 10 ng/ml IL-15, for 7 days with 10 ng/ml IL-15 and DMSO or for 7 days with 10 ng/ml IL-15 and 5 μM CHIR99021 prior to functional analysis. A , NK cells cultured in the conditions above were incubated with K562 cells at the indicated effector:target ratios followed by the addition of Caspase-3/7 Green Detection Reagent. FACS was used to determine the percentage of K562 cells undergoing apoptosis ( n = 15). Results are from 3 independent experiments and are shown as mean ± SEM. Paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01. NK cells cultured in the conditions above were incubated with K562 cells at a 1:1 ratio, and intracellular FACS was used to measure the percentage of CD3 − CD56 + NK cells positive for B , TNF and C , IFN-γ (right panel) ( n = 7). Results are from 4 independent experiments and are shown as mean ± SEM. Paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01. NK cells ( n = 3) cultured in the conditions above were incubated with D , A549 cells, E , SKOV-3 cells and F , PANC-1 cells expressing NucLight Red fluorescent protein at a 3:1 ratio with the addition of a control anti-CD20 antibody. Parallel assays with NK cells ( n = 3) cultured in the conditions above were performed with G , A549 cells, H, SKOV-3 cells and I, PANC-1 cells expressing NucLight Red fluorescent protein at a 3:1 ratio with the addition of either anti-Her2 or anti-EGFR antibodies. An IncuCyte Imaging System was used to measure target cell killing over 48 hours. Results are from 3 independent experiments and are shown as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons tests was used to determine significance.
Figure Legend Snippet: Inhibition of GSK3 with CHIR99021 enhances NK cell cytokine production and anti-tumor function in vitro CD3/19-depleted PBMCs from CMV seropositive donors were cultured overnight with 10 ng/ml IL-15, for 7 days with 10 ng/ml IL-15 and DMSO or for 7 days with 10 ng/ml IL-15 and 5 μM CHIR99021 prior to functional analysis. A , NK cells cultured in the conditions above were incubated with K562 cells at the indicated effector:target ratios followed by the addition of Caspase-3/7 Green Detection Reagent. FACS was used to determine the percentage of K562 cells undergoing apoptosis ( n = 15). Results are from 3 independent experiments and are shown as mean ± SEM. Paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01. NK cells cultured in the conditions above were incubated with K562 cells at a 1:1 ratio, and intracellular FACS was used to measure the percentage of CD3 − CD56 + NK cells positive for B , TNF and C , IFN-γ (right panel) ( n = 7). Results are from 4 independent experiments and are shown as mean ± SEM. Paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01. NK cells ( n = 3) cultured in the conditions above were incubated with D , A549 cells, E , SKOV-3 cells and F , PANC-1 cells expressing NucLight Red fluorescent protein at a 3:1 ratio with the addition of a control anti-CD20 antibody. Parallel assays with NK cells ( n = 3) cultured in the conditions above were performed with G , A549 cells, H, SKOV-3 cells and I, PANC-1 cells expressing NucLight Red fluorescent protein at a 3:1 ratio with the addition of either anti-Her2 or anti-EGFR antibodies. An IncuCyte Imaging System was used to measure target cell killing over 48 hours. Results are from 3 independent experiments and are shown as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons tests was used to determine significance.

Techniques Used: Inhibition, In Vitro, Cell Culture, Functional Assay, Incubation, FACS, Expressing, Imaging

NK cells expanded with CHIR99021 exhibit elevated expression of transcription factors associated with late-stage NK cell maturation CD3/19-depleted PBMCs from CMV seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and either DMSO or 5 μM CHIR99021 ( n = 9). CD3 − CD56 + NK cells were then isolated from each culture condition and analyzed by A , qRT-PCR for expression of TBX21 , ZEB2 , PRDM1A , PRDM1B and EOMES mRNA. Shown are cumulative data of relative fold expression values of cells cultured with CHIR99021 relative to cells cultured with DMSO. All data are normalized against ACTB . B , CD3 − CD56 + NK cells from these cultures were also analyzed by intracellular staining and FACS for expression levels of TBX21, ZEB2, BLIMP-1 and EOMES (as determined by mean fluorescence intensity). Shown are cumulative relative fold expression values for each transcription factor in cells cultured with CHIR99021 relative to cells cultured with DMSO. Results are from 2 independent experiments. Data is shown as mean ± SEM, and paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Figure Legend Snippet: NK cells expanded with CHIR99021 exhibit elevated expression of transcription factors associated with late-stage NK cell maturation CD3/19-depleted PBMCs from CMV seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and either DMSO or 5 μM CHIR99021 ( n = 9). CD3 − CD56 + NK cells were then isolated from each culture condition and analyzed by A , qRT-PCR for expression of TBX21 , ZEB2 , PRDM1A , PRDM1B and EOMES mRNA. Shown are cumulative data of relative fold expression values of cells cultured with CHIR99021 relative to cells cultured with DMSO. All data are normalized against ACTB . B , CD3 − CD56 + NK cells from these cultures were also analyzed by intracellular staining and FACS for expression levels of TBX21, ZEB2, BLIMP-1 and EOMES (as determined by mean fluorescence intensity). Shown are cumulative relative fold expression values for each transcription factor in cells cultured with CHIR99021 relative to cells cultured with DMSO. Results are from 2 independent experiments. Data is shown as mean ± SEM, and paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Techniques Used: Expressing, Cell Culture, Isolation, Quantitative RT-PCR, Staining, FACS, Fluorescence

Addition of CHIR99021 to NK cells expanded with autologous monocytes enriches for NK cells with a mature phenotype CD3/19-depleted PBMCs from CMV seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and either DMSO or 5 μM CHIR99021. A , Shown is fold NK cell expansion after 7 days in each culture condition relative to day 0 ( n = 15). B , Representative FACS scatter plots of CD57 and NKG2C expression on gated CD3 − CD56 + NK cells from before and after culture (upper panel). Cumulative ( n = 15) data showing the percentages of CD57 + NK cells and the mean fluorescence intensity of CD57 gated on CD57 + NK cells as well as the percentages of adaptive CD57 + NKG2C + NK cells within the total CD3 − CD56 + NK cell population before and after culture (lower panel). Results are from 5 independent experiments. C , Representative dot plots or FACS histogram plots for each receptor or cytotoxic granule component on gated CD3 − CD56 + NK cells from each culture condition (upper panel). Cumulative ( n = 6) data showing the relative median fluorescence intensity for each receptor or cytotoxic granule component on gated CD3 − CD56 + NK cells from DMSO and CHIR99021 cultures relative to the pre-culture phenotype (lower panel). All results are from 3–5 independent experiments. All cumulative data is shown as mean ± SEM, and paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Figure Legend Snippet: Addition of CHIR99021 to NK cells expanded with autologous monocytes enriches for NK cells with a mature phenotype CD3/19-depleted PBMCs from CMV seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and either DMSO or 5 μM CHIR99021. A , Shown is fold NK cell expansion after 7 days in each culture condition relative to day 0 ( n = 15). B , Representative FACS scatter plots of CD57 and NKG2C expression on gated CD3 − CD56 + NK cells from before and after culture (upper panel). Cumulative ( n = 15) data showing the percentages of CD57 + NK cells and the mean fluorescence intensity of CD57 gated on CD57 + NK cells as well as the percentages of adaptive CD57 + NKG2C + NK cells within the total CD3 − CD56 + NK cell population before and after culture (lower panel). Results are from 5 independent experiments. C , Representative dot plots or FACS histogram plots for each receptor or cytotoxic granule component on gated CD3 − CD56 + NK cells from each culture condition (upper panel). Cumulative ( n = 6) data showing the relative median fluorescence intensity for each receptor or cytotoxic granule component on gated CD3 − CD56 + NK cells from DMSO and CHIR99021 cultures relative to the pre-culture phenotype (lower panel). All results are from 3–5 independent experiments. All cumulative data is shown as mean ± SEM, and paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Techniques Used: Cell Culture, FACS, Expressing, Fluorescence

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Article Snippet: .. To isolate peripheral blood mononuclear Cells (PBMC), the red blood cells and platelets were removed with PBMC isolation kits according to the manufacturer's instruction (AllCells, Alameda, CA, USA). .. For T cell separation and activation, the acquired cell pellet was re-suspended in media X-VIVO15 and cultured.

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Article Snippet: .. Peripheral blood mononuclear cells (PBMCs) and positively selected primary normal human CD19+ B cells, CD4+ T cells, and CD14+ monocytes were purchased (AllCells, Emeryville, CA) or were isolated in-house (LeukoPak [StemCell and Physicians Plasma Alliance]). ..

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Article Title: GSK 3 inhibition drives maturation of NK cells and enhances their antitumor activity
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    AllCells LLC pbmcs
    Inference and effect of barcode multiplets in single-cell ATAC-seq data. (a) Default t-SNE depiction of public <t>scATAC-seq</t> <t>PBMC</t> 5k dataset. Colors represent cluster annotations from the automated CellRanger output. (b) Quantification of barcodes affected by barcode multiplets for the same dataset (identified by bap). (c) Depiction of two multiplets each composed of 9 oligonucleotide barcodes. Barcodes in each multiplet share a long common subsequence, denoted in black. (d) Visualization of two barcode multiplets from (c) in t-SNE coordinates. (e) Visualization of all implicated barcode multiplets from this dataset. The zoomed panel shows a small group of cells affected by five multiplets, indicated by color. (f) Empirical distribution of the mean restricted longest common subsequence (rLCS) per multiplet. A cutoff of 6 was used to determine either of the two classes of barcode multiplets. (g) Percent difference of the mean log2 fragments between pairs of barcodes within a multiplet. The reported p-value is from a two-sided Kolmogorov–Smirnov test. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. (h) Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs.
    Pbmcs, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AllCells LLC adcc assay human peripheral blood mononuclear cells pbmc
    <t>ADCC</t> of Y-443 and Fc-mutated antibodies against MDA-MB-231 cells. MDA-MB-231 cells pre-labeled with Calcein AM were incubated with different concentrations of Y-443 or the Fc mutants, followed by addition of <t>PBMC</t> effector cells at a ratio of 1:50. The cell mixture was incubated for 4 hours at 37°C, and Calcein AM intensity in cells was detected by Acumen eX3 (TTP labtech). The results are the mean ± S.D. of dead cell ratio.
    Adcc Assay Human Peripheral Blood Mononuclear Cells Pbmc, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AllCells LLC in vitro pd 1 receptor occupancy pbmcs
    Sintilimab showed in vitro and in vivo higher levels of <t>PD-1</t> receptor occupancy. Human <t>PBMC</t> were stimulated to express PD-1 before incubation with sintilimab, MDX-1106 or MK-3475. Flow cytometry results showing proportions of CD3+ T cells that bind with different anti-PD-1 mAbs (a) and the mean fluorescence intensity of PD-1 (b). Data are expressed as the means ± SE of three independent experiments. (c) The effects of anti-PD-1 mAbs on mixed lymphocyte reaction (MLR) response. CD4+ T cells isolated from human PBMC were co-cultured with mature monocyte-derived dendritic cells at a ratio of 10:1 in the presence of different concentrations of anti-PD-1 mAbs. Twelve hours later, unbound mAbs was removed. Cells were co-cultured for 4 more days and the concentration of IL-2 in cultural supernatant was detected by Cisbio kit. In NOG mice reconstituted with human immune cells, PD-1 receptor occupancy on circulating CD3+ T cells 24 h (d) and 72 h (e) after anti-PD-1 mAbs intraperitoneal injection at doses of 1, 3 and 10 mg/kg ( n ≥ 3 mice/group). (f) Mean (± SE) serum concentration-time profiles following a single IV administration of 10 mg/kg sintilimab, MDX-1106 or MK-3475 to hPD-1 knock-in mice (n = 3 animals per group).
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    Inference and effect of barcode multiplets in single-cell ATAC-seq data. (a) Default t-SNE depiction of public scATAC-seq PBMC 5k dataset. Colors represent cluster annotations from the automated CellRanger output. (b) Quantification of barcodes affected by barcode multiplets for the same dataset (identified by bap). (c) Depiction of two multiplets each composed of 9 oligonucleotide barcodes. Barcodes in each multiplet share a long common subsequence, denoted in black. (d) Visualization of two barcode multiplets from (c) in t-SNE coordinates. (e) Visualization of all implicated barcode multiplets from this dataset. The zoomed panel shows a small group of cells affected by five multiplets, indicated by color. (f) Empirical distribution of the mean restricted longest common subsequence (rLCS) per multiplet. A cutoff of 6 was used to determine either of the two classes of barcode multiplets. (g) Percent difference of the mean log2 fragments between pairs of barcodes within a multiplet. The reported p-value is from a two-sided Kolmogorov–Smirnov test. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. (h) Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs.

    Journal: bioRxiv

    Article Title: Inference and effects of barcode multiplets in droplet-based single-cell assays

    doi: 10.1101/824003

    Figure Lengend Snippet: Inference and effect of barcode multiplets in single-cell ATAC-seq data. (a) Default t-SNE depiction of public scATAC-seq PBMC 5k dataset. Colors represent cluster annotations from the automated CellRanger output. (b) Quantification of barcodes affected by barcode multiplets for the same dataset (identified by bap). (c) Depiction of two multiplets each composed of 9 oligonucleotide barcodes. Barcodes in each multiplet share a long common subsequence, denoted in black. (d) Visualization of two barcode multiplets from (c) in t-SNE coordinates. (e) Visualization of all implicated barcode multiplets from this dataset. The zoomed panel shows a small group of cells affected by five multiplets, indicated by color. (f) Empirical distribution of the mean restricted longest common subsequence (rLCS) per multiplet. A cutoff of 6 was used to determine either of the two classes of barcode multiplets. (g) Percent difference of the mean log2 fragments between pairs of barcodes within a multiplet. The reported p-value is from a two-sided Kolmogorov–Smirnov test. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. (h) Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs.

    Article Snippet: Profiling PBMCs using 10x scATAC-seq For 10x scATAC-seq experiments with PBMCs (PB003F, Allcells), frozen cells were quickly thawed in a 37°C water bath for about 30s and transferred to a 15 mL tube.

    Techniques: Chromatin Immunoprecipitation

    Supporting information for Figure 3 . (a) Quantification of barcodes affected by barcode multiplets for the PBMC dataset generated with this work (“This Study”). (b) Percentage of barcode multiplets identified for different numbers of input barcodes (see Methods ). (c) Visualization of seven additional barcode multiplets from the Public dataset. (d) Proportion of bead pairs occurring in the same chromatin accessibility-defined Louvain cluster compared to a permuted background. Error bars represent standard error of mean over 100 permutations per dataset. (e) Downsampling analysis of the dataset generated in this work (“This Study”). Barcode multiplets were examined at downsampled intervals from 10%-90% by units of 10%. The highlighted sample represents 40% downsampling and corresponds to a median 10,000 fragments detected per barcode. At all downsampled thresholds, we detected 0 pairs that were not present in the 100% sample. (f) Distribution of the restricted longest common subsequence (rLCS) for 1,000,000 randomly-sampled barcode pairs in the 10x barcode universe. A threshold at 6 is drawn for use in other analyses.

    Journal: bioRxiv

    Article Title: Inference and effects of barcode multiplets in droplet-based single-cell assays

    doi: 10.1101/824003

    Figure Lengend Snippet: Supporting information for Figure 3 . (a) Quantification of barcodes affected by barcode multiplets for the PBMC dataset generated with this work (“This Study”). (b) Percentage of barcode multiplets identified for different numbers of input barcodes (see Methods ). (c) Visualization of seven additional barcode multiplets from the Public dataset. (d) Proportion of bead pairs occurring in the same chromatin accessibility-defined Louvain cluster compared to a permuted background. Error bars represent standard error of mean over 100 permutations per dataset. (e) Downsampling analysis of the dataset generated in this work (“This Study”). Barcode multiplets were examined at downsampled intervals from 10%-90% by units of 10%. The highlighted sample represents 40% downsampling and corresponds to a median 10,000 fragments detected per barcode. At all downsampled thresholds, we detected 0 pairs that were not present in the 100% sample. (f) Distribution of the restricted longest common subsequence (rLCS) for 1,000,000 randomly-sampled barcode pairs in the 10x barcode universe. A threshold at 6 is drawn for use in other analyses.

    Article Snippet: Profiling PBMCs using 10x scATAC-seq For 10x scATAC-seq experiments with PBMCs (PB003F, Allcells), frozen cells were quickly thawed in a 37°C water bath for about 30s and transferred to a 15 mL tube.

    Techniques: Generated

    Cell surface expression of SAIL in CLL, AML and MM patient samples and normal BMMC and PBMC controls. ( a ) Three CLL specimens analyzed by flow cytometry. CLL cells were identified as CD19/CD5 double-positive cells. The histograms present SAIL (filled) and isotype control (open) staining in the live-cell and the CLL population. ( b ) Flow cytometry analysis of three AML specimens. SAIL expression is assessed in live-cells, CD33-positive and CD34-positive cells. ( c ) Flow cytometry analysis of three MM specimens. CD38 high cells with CD56 expression were gated for MM cells. SAIL expression is assessed in the live-cell and the MM population. ( d and e ) Flow cytometry analysis of SAIL expression in BMMC ( d ) and PBMC ( e ) via co-staining with CD19, CD3, CD14, CD56, CD33, CD34 and a cocktail of lineage (LN) markers. Numbers in histograms are median-fluorescence-intensity fold-change values relative to the isotype control. Three and two representative examples are shown for the tumor and normal samples, respectively.

    Journal: Blood Cancer Journal

    Article Title: A novel antibody–drug conjugate targeting SAIL for the treatment of hematologic malignancies

    doi: 10.1038/bcj.2015.39

    Figure Lengend Snippet: Cell surface expression of SAIL in CLL, AML and MM patient samples and normal BMMC and PBMC controls. ( a ) Three CLL specimens analyzed by flow cytometry. CLL cells were identified as CD19/CD5 double-positive cells. The histograms present SAIL (filled) and isotype control (open) staining in the live-cell and the CLL population. ( b ) Flow cytometry analysis of three AML specimens. SAIL expression is assessed in live-cells, CD33-positive and CD34-positive cells. ( c ) Flow cytometry analysis of three MM specimens. CD38 high cells with CD56 expression were gated for MM cells. SAIL expression is assessed in the live-cell and the MM population. ( d and e ) Flow cytometry analysis of SAIL expression in BMMC ( d ) and PBMC ( e ) via co-staining with CD19, CD3, CD14, CD56, CD33, CD34 and a cocktail of lineage (LN) markers. Numbers in histograms are median-fluorescence-intensity fold-change values relative to the isotype control. Three and two representative examples are shown for the tumor and normal samples, respectively.

    Article Snippet: Fresh specimens from acute myeloid leukemia (AML) and multiple myeloma (MM) patients and normal peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) from nondiseased donors were acquired from AllCells (Emeryville, CA, USA).

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining, Fluorescence

    Proteomic identification of SAIL in hematologic malignancies. Expression of SAIL was analyzed in 14 AML, 40 CLL and 33 MM patient specimens, as well as in 21 normal BMMC and 20 normal PBMC controls. The relative quantitative protein abundance was determined using mass spectrometry-based spectral counting. Raw spectral counts were calculated as % NSAF.

    Journal: Blood Cancer Journal

    Article Title: A novel antibody–drug conjugate targeting SAIL for the treatment of hematologic malignancies

    doi: 10.1038/bcj.2015.39

    Figure Lengend Snippet: Proteomic identification of SAIL in hematologic malignancies. Expression of SAIL was analyzed in 14 AML, 40 CLL and 33 MM patient specimens, as well as in 21 normal BMMC and 20 normal PBMC controls. The relative quantitative protein abundance was determined using mass spectrometry-based spectral counting. Raw spectral counts were calculated as % NSAF.

    Article Snippet: Fresh specimens from acute myeloid leukemia (AML) and multiple myeloma (MM) patients and normal peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) from nondiseased donors were acquired from AllCells (Emeryville, CA, USA).

    Techniques: Expressing, Mass Spectrometry

    ADCC of Y-443 and Fc-mutated antibodies against MDA-MB-231 cells. MDA-MB-231 cells pre-labeled with Calcein AM were incubated with different concentrations of Y-443 or the Fc mutants, followed by addition of PBMC effector cells at a ratio of 1:50. The cell mixture was incubated for 4 hours at 37°C, and Calcein AM intensity in cells was detected by Acumen eX3 (TTP labtech). The results are the mean ± S.D. of dead cell ratio.

    Journal: PLoS ONE

    Article Title: Fc engineering of anti-Nectin-2 antibody improved thrombocytopenic adverse event in monkey

    doi: 10.1371/journal.pone.0196422

    Figure Lengend Snippet: ADCC of Y-443 and Fc-mutated antibodies against MDA-MB-231 cells. MDA-MB-231 cells pre-labeled with Calcein AM were incubated with different concentrations of Y-443 or the Fc mutants, followed by addition of PBMC effector cells at a ratio of 1:50. The cell mixture was incubated for 4 hours at 37°C, and Calcein AM intensity in cells was detected by Acumen eX3 (TTP labtech). The results are the mean ± S.D. of dead cell ratio.

    Article Snippet: ADCC assay Human peripheral blood mononuclear cells (PBMC) purchased from AllCells, LLC and were cultured in RPMI1640 medium containing 10% fetal bovine serum, 0.1 nM human IL-2 (DIACLONE Research) and 55 μM 2-mercaptoethanol for 24 hours.

    Techniques: Multiple Displacement Amplification, Labeling, Incubation

    Sintilimab showed in vitro and in vivo higher levels of PD-1 receptor occupancy. Human PBMC were stimulated to express PD-1 before incubation with sintilimab, MDX-1106 or MK-3475. Flow cytometry results showing proportions of CD3+ T cells that bind with different anti-PD-1 mAbs (a) and the mean fluorescence intensity of PD-1 (b). Data are expressed as the means ± SE of three independent experiments. (c) The effects of anti-PD-1 mAbs on mixed lymphocyte reaction (MLR) response. CD4+ T cells isolated from human PBMC were co-cultured with mature monocyte-derived dendritic cells at a ratio of 10:1 in the presence of different concentrations of anti-PD-1 mAbs. Twelve hours later, unbound mAbs was removed. Cells were co-cultured for 4 more days and the concentration of IL-2 in cultural supernatant was detected by Cisbio kit. In NOG mice reconstituted with human immune cells, PD-1 receptor occupancy on circulating CD3+ T cells 24 h (d) and 72 h (e) after anti-PD-1 mAbs intraperitoneal injection at doses of 1, 3 and 10 mg/kg ( n ≥ 3 mice/group). (f) Mean (± SE) serum concentration-time profiles following a single IV administration of 10 mg/kg sintilimab, MDX-1106 or MK-3475 to hPD-1 knock-in mice (n = 3 animals per group).

    Journal: mAbs

    Article Title: Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit

    doi: 10.1080/19420862.2019.1654303

    Figure Lengend Snippet: Sintilimab showed in vitro and in vivo higher levels of PD-1 receptor occupancy. Human PBMC were stimulated to express PD-1 before incubation with sintilimab, MDX-1106 or MK-3475. Flow cytometry results showing proportions of CD3+ T cells that bind with different anti-PD-1 mAbs (a) and the mean fluorescence intensity of PD-1 (b). Data are expressed as the means ± SE of three independent experiments. (c) The effects of anti-PD-1 mAbs on mixed lymphocyte reaction (MLR) response. CD4+ T cells isolated from human PBMC were co-cultured with mature monocyte-derived dendritic cells at a ratio of 10:1 in the presence of different concentrations of anti-PD-1 mAbs. Twelve hours later, unbound mAbs was removed. Cells were co-cultured for 4 more days and the concentration of IL-2 in cultural supernatant was detected by Cisbio kit. In NOG mice reconstituted with human immune cells, PD-1 receptor occupancy on circulating CD3+ T cells 24 h (d) and 72 h (e) after anti-PD-1 mAbs intraperitoneal injection at doses of 1, 3 and 10 mg/kg ( n ≥ 3 mice/group). (f) Mean (± SE) serum concentration-time profiles following a single IV administration of 10 mg/kg sintilimab, MDX-1106 or MK-3475 to hPD-1 knock-in mice (n = 3 animals per group).

    Article Snippet: In vitro PD-1 receptor occupancy PBMCs (AllCells, Alameda, California, USA) were activated by human dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific) for 48 h to induce PD-1 expression and were then incubated with sintilimab, MDX-1106, or MK-3475 at a concentration of 150 ng/μl.

    Techniques: In Vitro, In Vivo, Incubation, Flow Cytometry, Cytometry, Fluorescence, Isolation, Cell Culture, Derivative Assay, Concentration Assay, Mouse Assay, Injection, Knock-In