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Lentiviral vector delivery of hIL-7 promotes homeostatic proliferation of adoptively transferred human T cells in Rag2-/-γc-/- mice. a. Serum concentrations of hIL-7 detected by ELISA three weeks after intravenous administration of 9×10 7 or 1.7×10 8 IU of lentivirus expressing either luciferase or hIL-7. b. The percentage of CD3+, CD4+ or CD8+ T cells of live splenocytes following one week post transfer of 2×10 7 <t>CFSE</t> labeled human <t>PBMCs</t> into Rag2-/-γc-/- mice from A. c. Average mean fluorescence intensity (MFI) of CFSE measured by flow cytometry in T-cell subsets quantified in B. Four mice were used per group, and the average and SEM are shown. d. Representative histograms showing CFSE loss by CD3+, CD4+ or CD8+ adoptively transferred T cells from mice receiving the control vector, low dose hIL-7 or high dose hIL-7.
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1) Product Images from "Lentiviral Vector Delivery of Human Interleukin-7 (hIL-7) to Human Immune System (HIS) Mice Expands T Lymphocyte Populations"

Article Title: Lentiviral Vector Delivery of Human Interleukin-7 (hIL-7) to Human Immune System (HIS) Mice Expands T Lymphocyte Populations

Journal: PLoS ONE

doi: 10.1371/journal.pone.0012009

Lentiviral vector delivery of hIL-7 promotes homeostatic proliferation of adoptively transferred human T cells in Rag2-/-γc-/- mice. a. Serum concentrations of hIL-7 detected by ELISA three weeks after intravenous administration of 9×10 7 or 1.7×10 8 IU of lentivirus expressing either luciferase or hIL-7. b. The percentage of CD3+, CD4+ or CD8+ T cells of live splenocytes following one week post transfer of 2×10 7 CFSE labeled human PBMCs into Rag2-/-γc-/- mice from A. c. Average mean fluorescence intensity (MFI) of CFSE measured by flow cytometry in T-cell subsets quantified in B. Four mice were used per group, and the average and SEM are shown. d. Representative histograms showing CFSE loss by CD3+, CD4+ or CD8+ adoptively transferred T cells from mice receiving the control vector, low dose hIL-7 or high dose hIL-7.
Figure Legend Snippet: Lentiviral vector delivery of hIL-7 promotes homeostatic proliferation of adoptively transferred human T cells in Rag2-/-γc-/- mice. a. Serum concentrations of hIL-7 detected by ELISA three weeks after intravenous administration of 9×10 7 or 1.7×10 8 IU of lentivirus expressing either luciferase or hIL-7. b. The percentage of CD3+, CD4+ or CD8+ T cells of live splenocytes following one week post transfer of 2×10 7 CFSE labeled human PBMCs into Rag2-/-γc-/- mice from A. c. Average mean fluorescence intensity (MFI) of CFSE measured by flow cytometry in T-cell subsets quantified in B. Four mice were used per group, and the average and SEM are shown. d. Representative histograms showing CFSE loss by CD3+, CD4+ or CD8+ adoptively transferred T cells from mice receiving the control vector, low dose hIL-7 or high dose hIL-7.

Techniques Used: Plasmid Preparation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Expressing, Luciferase, Labeling, Fluorescence, Flow Cytometry, Cytometry

2) Product Images from "Real Time Assays for Quantifying Cytotoxicity with Single Cell Resolution"

Article Title: Real Time Assays for Quantifying Cytotoxicity with Single Cell Resolution

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066739

Cell-array ADCC assays. The scale bar in each image represents 100 µm. (a) The bright field images show a mixture of indistinguishable targeted cells and effector cells (PBMCs) after 16 h. (b) The Jeko-1 target cells were prestained with a Cell Tracker dye, allowing their facile detection using fluorescence microscopy. (c) The dead cells were stained red using PI and counted using Image-J software or manually. (d) A merged image allows the ratio of living (blue) to dead (purple) targeted cells to be determined. Red cells with no overlapping blue stain indicated dead effector cells, which were not counted. Cellular microarray ADCC results are shown for the dose-dependent killing of (e) Jeko-1 cells and (f) primary B-chronic lymphobatic leukemia cells with α-CD20 after 16 h. The error bars indicate the standard deviation of three replicate experiments.
Figure Legend Snippet: Cell-array ADCC assays. The scale bar in each image represents 100 µm. (a) The bright field images show a mixture of indistinguishable targeted cells and effector cells (PBMCs) after 16 h. (b) The Jeko-1 target cells were prestained with a Cell Tracker dye, allowing their facile detection using fluorescence microscopy. (c) The dead cells were stained red using PI and counted using Image-J software or manually. (d) A merged image allows the ratio of living (blue) to dead (purple) targeted cells to be determined. Red cells with no overlapping blue stain indicated dead effector cells, which were not counted. Cellular microarray ADCC results are shown for the dose-dependent killing of (e) Jeko-1 cells and (f) primary B-chronic lymphobatic leukemia cells with α-CD20 after 16 h. The error bars indicate the standard deviation of three replicate experiments.

Techniques Used: Fluorescence, Microscopy, Staining, Software, Microarray, Standard Deviation

3) Product Images from "Single-cell multimodal profiling of proteins and chromatin accessibility using PHAGE-ATAC"

Article Title: Single-cell multimodal profiling of proteins and chromatin accessibility using PHAGE-ATAC

Journal: bioRxiv

doi: 10.1101/2020.10.01.322420

Multimodal single-cell analysis of human PBMCs using PHAGE-ATAC (A) Two-dimensional joint embedding of scRNA-seq profiles from PBMCs from published CITE-seq ( Stoeckius et al., 2017 ) and of scATAC-seq profiles from PBMCs generated by PHAGE-ATAC, colored by the measured RNA level from CITE-Seq (top panels) or by gene activity scores from PHAGE-ATAC (bottom panels) ( Methods ). (B) , (C) PHAGE-ATAC gating by phage staining highlights cell type specific loci. (B) PDT count-based classification of CD4 + and CD8 + T cells. PDT counts (CLR transformed) of CD8 (y axis) and CD4 (x axis) in each cell (dots). Red boxes: gates for CD4+ and CD8+ cells. (C) Average fold change (x axis, log 2 ) and associated significance (y axis, −log 10 (P-value) for each gene activity comparing between PDT-classified CD4 and CD8 T cells shown in B. Known bona fide markers of either CD4 or CD8 T cells are marked. (D) Negative control. Embedding of PHAGE-ATAC data as in A, colored by anti-EGFP pNb PDT. (E) Distribution of phage counts (y axis, log 10 ) for each cell barcode for each assayed nanobody (x axis).
Figure Legend Snippet: Multimodal single-cell analysis of human PBMCs using PHAGE-ATAC (A) Two-dimensional joint embedding of scRNA-seq profiles from PBMCs from published CITE-seq ( Stoeckius et al., 2017 ) and of scATAC-seq profiles from PBMCs generated by PHAGE-ATAC, colored by the measured RNA level from CITE-Seq (top panels) or by gene activity scores from PHAGE-ATAC (bottom panels) ( Methods ). (B) , (C) PHAGE-ATAC gating by phage staining highlights cell type specific loci. (B) PDT count-based classification of CD4 + and CD8 + T cells. PDT counts (CLR transformed) of CD8 (y axis) and CD4 (x axis) in each cell (dots). Red boxes: gates for CD4+ and CD8+ cells. (C) Average fold change (x axis, log 2 ) and associated significance (y axis, −log 10 (P-value) for each gene activity comparing between PDT-classified CD4 and CD8 T cells shown in B. Known bona fide markers of either CD4 or CD8 T cells are marked. (D) Negative control. Embedding of PHAGE-ATAC data as in A, colored by anti-EGFP pNb PDT. (E) Distribution of phage counts (y axis, log 10 ) for each cell barcode for each assayed nanobody (x axis).

Techniques Used: Single-cell Analysis, Generated, Activity Assay, Staining, Transformation Assay, Significance Assay, Negative Control

Optimization of fixation and lysis conditions for PHAGE-ATAC using PBMCs (A) Binding of generated anti-CD4 phage nanobodies to PBMCs under indicated conditions. Two different formaldehyde concentrations as well as various depicted lysis buffers were used. Phage binding is reflected by Alexa Fluor 647 fluorescent signal intensity. (B) Histogram of data in (A) .
Figure Legend Snippet: Optimization of fixation and lysis conditions for PHAGE-ATAC using PBMCs (A) Binding of generated anti-CD4 phage nanobodies to PBMCs under indicated conditions. Two different formaldehyde concentrations as well as various depicted lysis buffers were used. Phage binding is reflected by Alexa Fluor 647 fluorescent signal intensity. (B) Histogram of data in (A) .

Techniques Used: Lysis, Binding Assay, Generated

Sample multiplexing using hashtag phages (A) Validation of phage hashtag binding. Flow cytometry of anti-CD8 hashtag phages bound (Alexa Fluor 647 fluorescent signal, x axis) to lymphocytes gated via flow cytometry of phage-stained PBMCs (as shown in Supp. Fig. 8A ). (B) Cell type identification. Two-dimensional embedding of hashed CD8 T cells analyzed by PHAGE-ATAC, colored by cell type annotation. (C) Pseudobulk chromatin accessibility track plots for CD8, CD3 and MS4A1 ( CD20 ) loci across identified cell types. (D) Embedding as in B with cells colored by CD8 hashtag PDTs. (E),(F) Distribution of maximal CD8 PDT density (E, y axis) or unique chromatin fragments (F, y axis) for each cell barcode in CD8 - (B cell 1 and B cell 2) and CD8 + (non-B cell) cells (x axis) (Mann-Whitney two-tailed, ***p
Figure Legend Snippet: Sample multiplexing using hashtag phages (A) Validation of phage hashtag binding. Flow cytometry of anti-CD8 hashtag phages bound (Alexa Fluor 647 fluorescent signal, x axis) to lymphocytes gated via flow cytometry of phage-stained PBMCs (as shown in Supp. Fig. 8A ). (B) Cell type identification. Two-dimensional embedding of hashed CD8 T cells analyzed by PHAGE-ATAC, colored by cell type annotation. (C) Pseudobulk chromatin accessibility track plots for CD8, CD3 and MS4A1 ( CD20 ) loci across identified cell types. (D) Embedding as in B with cells colored by CD8 hashtag PDTs. (E),(F) Distribution of maximal CD8 PDT density (E, y axis) or unique chromatin fragments (F, y axis) for each cell barcode in CD8 - (B cell 1 and B cell 2) and CD8 + (non-B cell) cells (x axis) (Mann-Whitney two-tailed, ***p

Techniques Used: Multiplexing, Binding Assay, Flow Cytometry, Staining, MANN-WHITNEY, Two Tailed Test

Validation of PAC-tagged anti-CD4, anti-CD8 and anti-CD16 nanobody-displaying phages (A) Flow cytometry gating strategy for analyzed phage-stained PBMCs. (B) Flow cytometry-based binding assessment of indicated surface marker-recognizing phage nanobodies to gated lymphocyte and monocyte populations, anti-EGFP pNb was used as negative control. (C) Comparison of PBMCs stained with a well-characterized anti-CD4 antibody or generated anti-CD4 phage nanobody. Phage binding is reflected by Alexa Fluor 647 fluorescent signal intensity.
Figure Legend Snippet: Validation of PAC-tagged anti-CD4, anti-CD8 and anti-CD16 nanobody-displaying phages (A) Flow cytometry gating strategy for analyzed phage-stained PBMCs. (B) Flow cytometry-based binding assessment of indicated surface marker-recognizing phage nanobodies to gated lymphocyte and monocyte populations, anti-EGFP pNb was used as negative control. (C) Comparison of PBMCs stained with a well-characterized anti-CD4 antibody or generated anti-CD4 phage nanobody. Phage binding is reflected by Alexa Fluor 647 fluorescent signal intensity.

Techniques Used: Flow Cytometry, Staining, Binding Assay, Marker, Negative Control, Generated

4) Product Images from "Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit"

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

Journal: mAbs

doi: 10.1080/19420862.2019.1654303

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).
Figure Legend 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).

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

5) Product Images from "Inference and effects of barcode multiplets in droplet-based single-cell assays"

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

Journal: Nature Communications

doi: 10.1038/s41467-020-14667-5

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. The exact p -value is lower than machine precision. Analysis represents n = 5205 barcodes over 1 experimental replicate. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range. h Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs. Source data are available in the Source Data file.
Figure Legend 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. The exact p -value is lower than machine precision. Analysis represents n = 5205 barcodes over 1 experimental replicate. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range. h Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs. Source data are available in the Source Data file.

Techniques Used: Chromatin Immunoprecipitation

6) Product Images from "Inhibition of HIV Replication by Apolipoprotein A-I Binding Protein Targeting the Lipid Rafts"

Article Title: Inhibition of HIV Replication by Apolipoprotein A-I Binding Protein Targeting the Lipid Rafts

Journal: mBio

doi: 10.1128/mBio.02956-19

Anti-HIV effect of AIBP is reduced in cells from HLA-B*35 donors. (A) PHA-activated PBMCs from donors with HLA-B*35, HLA-B*57, and non-B*35,B*57 genotypes were infected with HIV-1 LAI and incubated in the presence or absence of recombinant AIBP. Virus replication was followed by analysis of RT activity. Results are presented for donors B*35/55 (B*35), B*51/57 (B*57), and B*27/38 (non-B*35,B*57). Results are presented as means ± SD of results from 5 replicates. *, P = 0.01; **, P
Figure Legend Snippet: Anti-HIV effect of AIBP is reduced in cells from HLA-B*35 donors. (A) PHA-activated PBMCs from donors with HLA-B*35, HLA-B*57, and non-B*35,B*57 genotypes were infected with HIV-1 LAI and incubated in the presence or absence of recombinant AIBP. Virus replication was followed by analysis of RT activity. Results are presented for donors B*35/55 (B*35), B*51/57 (B*57), and B*27/38 (non-B*35,B*57). Results are presented as means ± SD of results from 5 replicates. *, P = 0.01; **, P

Techniques Used: Infection, Incubation, Recombinant, Activity Assay

7) Product Images from "Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins"

Article Title: Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.01106

The functional responses of Treg cells are quantitatively sensitive to attenuated IL-2 signal. (A) Proliferation of Treg cells in response to select muteins from each class was measured in human PBMC assay and shown as percent Ki67-positive cells in CD25+ Foxp3+ CD4-gated Treg cells. Shown are data representative of four donors. (B) Proliferative responses measured as percent Ki67 positives of Treg, CD25+ Tconv, NK (CD56+ CD3–), and CD8 T cell gated subpopulations to increasing concentrations of wildtype or IL-2 muteins are compared in a total PBMC assay, for select muteins from each class. Shown are representative of data from four donors. (C) IL-2 mutein activity on induction of Foxp3 and CTLA4 in Treg cells are shown, represented as MFI of Foxp3 or CTLA4 in Foxp3-positive and CTLA4-positive gated Treg cell populations, respectively. (D) Treg phenotype before and after stimulation. Purified human Treg cells were stimulated with anti-CD3 and wildtype or IL-2 mutein at 66.7 nM for 3 days and analyzed for Foxp3 and CD25 expression. Day 0 unstimulated Treg cells analyzed at the same time to show baseline expression of these markers. Each color represents the mutein class or day 0: wildtype (black closed circles), class A (blue), class B (green), class C (red), and day 0 (black open circles). Shown are combined data from three different donors. ** represents p -value of 0.005 from a one-way ANOVA analysis. (E) Treg suppression assay. Purified human Treg cells that were pre-stimulated with anti-CD3 and IL-2 mutein were co-cultured with purified CD8 T cells from an unmatched donor at various ratios. CD8 T cells were stimulated with CD3+CD28 activation beads and Treg-mediated suppression was measured by activation marker induction on CD8 T cells on day 1. These values were converted to percent suppression, each point representing the average of values from two donors. Error bars indicate standard deviation.
Figure Legend Snippet: The functional responses of Treg cells are quantitatively sensitive to attenuated IL-2 signal. (A) Proliferation of Treg cells in response to select muteins from each class was measured in human PBMC assay and shown as percent Ki67-positive cells in CD25+ Foxp3+ CD4-gated Treg cells. Shown are data representative of four donors. (B) Proliferative responses measured as percent Ki67 positives of Treg, CD25+ Tconv, NK (CD56+ CD3–), and CD8 T cell gated subpopulations to increasing concentrations of wildtype or IL-2 muteins are compared in a total PBMC assay, for select muteins from each class. Shown are representative of data from four donors. (C) IL-2 mutein activity on induction of Foxp3 and CTLA4 in Treg cells are shown, represented as MFI of Foxp3 or CTLA4 in Foxp3-positive and CTLA4-positive gated Treg cell populations, respectively. (D) Treg phenotype before and after stimulation. Purified human Treg cells were stimulated with anti-CD3 and wildtype or IL-2 mutein at 66.7 nM for 3 days and analyzed for Foxp3 and CD25 expression. Day 0 unstimulated Treg cells analyzed at the same time to show baseline expression of these markers. Each color represents the mutein class or day 0: wildtype (black closed circles), class A (blue), class B (green), class C (red), and day 0 (black open circles). Shown are combined data from three different donors. ** represents p -value of 0.005 from a one-way ANOVA analysis. (E) Treg suppression assay. Purified human Treg cells that were pre-stimulated with anti-CD3 and IL-2 mutein were co-cultured with purified CD8 T cells from an unmatched donor at various ratios. CD8 T cells were stimulated with CD3+CD28 activation beads and Treg-mediated suppression was measured by activation marker induction on CD8 T cells on day 1. These values were converted to percent suppression, each point representing the average of values from two donors. Error bars indicate standard deviation.

Techniques Used: Functional Assay, PBMC Assay, Activity Assay, Purification, Expressing, Suppression Assay, Cell Culture, Activation Assay, Marker, Standard Deviation

8) Product Images from "Inference and effects of barcode multiplets in droplet-based single-cell assays"

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

Journal: bioRxiv

doi: 10.1101/824003

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Generated

9) Product Images from "Inference and effects of barcode multiplets in droplet-based single-cell assays"

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

Journal: bioRxiv

doi: 10.1101/824003

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Generated

10) Product Images from "Impaired gamma delta T cell-derived IL-17A and inflammasome activation during early respiratory syncytial virus infection in infants"

Article Title: Impaired gamma delta T cell-derived IL-17A and inflammasome activation during early respiratory syncytial virus infection in infants

Journal: Immunology and cell biology

doi: 10.1038/icb.2014.79

Impaired IL-17A and IFNγ responses in γδ T cells following inflammasome activation in human cord blood mononuclear cells Human CBMCs and adult PBMCs (2 × 10 5 cells/well) were treated in vitro with a RIG-I agonist (1 μg/ml, 48 hr). ( A ) γδ T cells were quantified in pre- and post-treatment samples by flow cytometry. ( B,C ) Following incubation, IL-17A and IFNγ iMFI was determined in γδ T cells by flow cytometry. Production of IL-1β ( D ), IL-6 ( E ), TNFα ( F ), and IL-18 ( G ) were measured in cell culture supernatants by multiplex or ELISA assay. ( H ) Expression of active caspase-1in CBMCs (black fill) vs. adult PBMCs (open) compared to FMO control (grey fill). Data are representative of four independent experiments. N=5 samples per group from individual donors per experiment. Data plotted as means ± SEM. * P
Figure Legend Snippet: Impaired IL-17A and IFNγ responses in γδ T cells following inflammasome activation in human cord blood mononuclear cells Human CBMCs and adult PBMCs (2 × 10 5 cells/well) were treated in vitro with a RIG-I agonist (1 μg/ml, 48 hr). ( A ) γδ T cells were quantified in pre- and post-treatment samples by flow cytometry. ( B,C ) Following incubation, IL-17A and IFNγ iMFI was determined in γδ T cells by flow cytometry. Production of IL-1β ( D ), IL-6 ( E ), TNFα ( F ), and IL-18 ( G ) were measured in cell culture supernatants by multiplex or ELISA assay. ( H ) Expression of active caspase-1in CBMCs (black fill) vs. adult PBMCs (open) compared to FMO control (grey fill). Data are representative of four independent experiments. N=5 samples per group from individual donors per experiment. Data plotted as means ± SEM. * P

Techniques Used: Activation Assay, In Vitro, Flow Cytometry, Cytometry, Incubation, Cell Culture, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Expressing

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Article Snippet: Co-cultures of UC-MSCs and PBMCs PBMCs obtained from patients with relapsing-remitting multiple sclerosis (RRMS, n=12, female=8, male=4), mean age 53.75 years old (49 years to 64 years old) and healthy donors (n=10, female=4, male=6), mean age 28.38 years old (22 years to 39 years old) were purchased from AllCells Inc (Emeryville, CA) or StemExpress (Placerville, CA).

Article Title: EGR2 is elevated and positively regulates inflammatory IFNγ production in lupus CD4+ T cells
Article Snippet: Human peripheral blood mononuclear cells (PBMCs) The PBMCs of human patients of lupus (n = 4, all female) and healthy controls (n = 4, 2 male and 2 female) were purchased directly from AllCells LLC (Alameda, CA, USA).

Flow Cytometry:

Article Title: Single-cell multimodal profiling of proteins and chromatin accessibility using PHAGE-ATAC
Article Snippet: PHAGE-ATAC workflowFor cell line “species mixing” experiment, culture medium was aspirated, cell lines were washed with PBS, harvested using Trypsin-EDTA 0.25% (Thermo Scientific), resuspended in DMEM containing 10% FBS, centrifuged, washed with PBS and resuspended in FC buffer. .. For PBMC and CD8 T cell experiments, cryopreserved PBMCs or CD8 T cells (AllCells) were thawed, washed in PBS and resuspended in cold Flow cytometry buffer (FC buffer; PBS containing 2% FBS). .. All centrifugation steps were carried out at 350g, 4min, 4°C unless stated otherwise.

Western Blot:

Article Title: Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results
Article Snippet: PBMCs were purchased from AllCells Inc. (Alameda, CA, USA). .. Cryopreserved PBMCs were resuspended in RPMI 1640 supplemented with 10% FBS at 37°C in accordance with the protocol provided by AllCells Inc. PBMCs from HTLV-1 WB-indeterminate pregnant women were obtained with informed consent. .. Blood clots from HTLV-1 WB-indeterminate blood donors were obtained in two different areas of HTLV-1 epidemiology, the Kanto Block Blood Center, in an area where HTLV-1 infection is not endemic, including the prefectures of Tokyo and Chiba, and the Kyushu Block Blood Center, in an area where HTLV-1 infection is not endemic, including the prefectures of Kyushu Island.

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    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.
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    Cell surface expression of SAIL in CLL, <t>AML</t> and MM patient samples and normal BMMC and <t>PBMC</t> 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.
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    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

    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

    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

    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