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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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1) 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

2) Product Images from "Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice"

Article Title: Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice

Journal: Blood

doi: 10.1182/blood-2012-04-422303

HIV-1 infection in humanized mice passively infused with b12-IgA or b12-IgG isotype. (A) Neutralization activity of anti-HIV human mAb b12-IgA. Reporter cell line TZM-bl cells that express CD4, CXCR4, CCR5, and a Tat-responsive reporter gene for luciferase were infected with 200 TCID 50 of replication-defective pseudovirus containing Env (SF162.LS) in the presence of various concentrations of anti-HIV monoclonal antibodies. Neutralization activity was measured by the reduction in luciferase reporter gene expression after a single round of pseudovirus infection in TZM-bl cells in triplicate. b12-IgG1 and the recombinant b12-IgA2 were compared at indicated concentrations. (B) Antibody level in circulation after passive transfer. NSG-hu mice were injected intravenously with various concentrations of purified b12-IgA2 (pIgA: mIgA = 1:1, mass ratio) or b12-IgG1 and the blood was collected after 4 hours. The plasma antibody concentrations were measured using ELISA. (n = 4-6, mean ± SEM). (C-D) Concentrations of b12 antibodies in plasma (C) or genital secretions (D) at the time of challenge. NSG-hu mice were injected intravenously with either 200 μg of b12-IgA2 or 20 μg of b12-IgG1 per mouse. Blood and genital secretions were collected after 4 hours. (E-I) Peripheral CD4 + T cell loss after HIV-1 challenge in NSG-hu mice injected with different b12 antibody isotypes; (E) purified human IgG/κ control antibody (hIgG/κ); (F) b12-IgA2 includes both monomeric and polymeric IgA as described in (B), (b12IgA2 [M+P]); (G) b12-IgG1; (H) b12-IgA2 monomer only (b12IgA2 [M]). Mice were challenged intravaginally with HIV-1 JR-CSF . (I) Average percent CD4 + T cells in CD3 T cells in PBMCs at each time points (n = 5-9). Statistical analysis was performed using unpaired t test. (* P = .0313 and *** P
Figure Legend Snippet: HIV-1 infection in humanized mice passively infused with b12-IgA or b12-IgG isotype. (A) Neutralization activity of anti-HIV human mAb b12-IgA. Reporter cell line TZM-bl cells that express CD4, CXCR4, CCR5, and a Tat-responsive reporter gene for luciferase were infected with 200 TCID 50 of replication-defective pseudovirus containing Env (SF162.LS) in the presence of various concentrations of anti-HIV monoclonal antibodies. Neutralization activity was measured by the reduction in luciferase reporter gene expression after a single round of pseudovirus infection in TZM-bl cells in triplicate. b12-IgG1 and the recombinant b12-IgA2 were compared at indicated concentrations. (B) Antibody level in circulation after passive transfer. NSG-hu mice were injected intravenously with various concentrations of purified b12-IgA2 (pIgA: mIgA = 1:1, mass ratio) or b12-IgG1 and the blood was collected after 4 hours. The plasma antibody concentrations were measured using ELISA. (n = 4-6, mean ± SEM). (C-D) Concentrations of b12 antibodies in plasma (C) or genital secretions (D) at the time of challenge. NSG-hu mice were injected intravenously with either 200 μg of b12-IgA2 or 20 μg of b12-IgG1 per mouse. Blood and genital secretions were collected after 4 hours. (E-I) Peripheral CD4 + T cell loss after HIV-1 challenge in NSG-hu mice injected with different b12 antibody isotypes; (E) purified human IgG/κ control antibody (hIgG/κ); (F) b12-IgA2 includes both monomeric and polymeric IgA as described in (B), (b12IgA2 [M+P]); (G) b12-IgG1; (H) b12-IgA2 monomer only (b12IgA2 [M]). Mice were challenged intravaginally with HIV-1 JR-CSF . (I) Average percent CD4 + T cells in CD3 T cells in PBMCs at each time points (n = 5-9). Statistical analysis was performed using unpaired t test. (* P = .0313 and *** P

Techniques Used: Infection, Mouse Assay, Neutralization, Activity Assay, Luciferase, Expressing, Recombinant, Injection, Purification, Enzyme-linked Immunosorbent Assay

3) Product Images from "Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice"

Article Title: Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice

Journal: Blood

doi: 10.1182/blood-2012-04-422303

HIV-1 infection in humanized mice passively infused with b12-IgA or b12-IgG isotype. (A) Neutralization activity of anti-HIV human mAb b12-IgA. Reporter cell line TZM-bl cells that express CD4, CXCR4, CCR5, and a Tat-responsive reporter gene for luciferase were infected with 200 TCID 50 of replication-defective pseudovirus containing Env (SF162.LS) in the presence of various concentrations of anti-HIV monoclonal antibodies. Neutralization activity was measured by the reduction in luciferase reporter gene expression after a single round of pseudovirus infection in TZM-bl cells in triplicate. b12-IgG1 and the recombinant b12-IgA2 were compared at indicated concentrations. (B) Antibody level in circulation after passive transfer. NSG-hu mice were injected intravenously with various concentrations of purified b12-IgA2 (pIgA: mIgA = 1:1, mass ratio) or b12-IgG1 and the blood was collected after 4 hours. The plasma antibody concentrations were measured using ELISA. (n = 4-6, mean ± SEM). (C-D) Concentrations of b12 antibodies in plasma (C) or genital secretions (D) at the time of challenge. NSG-hu mice were injected intravenously with either 200 μg of b12-IgA2 or 20 μg of b12-IgG1 per mouse. Blood and genital secretions were collected after 4 hours. (E-I) Peripheral CD4 + T cell loss after HIV-1 challenge in NSG-hu mice injected with different b12 antibody isotypes; (E) purified human IgG/κ control antibody (hIgG/κ); (F) b12-IgA2 includes both monomeric and polymeric IgA as described in (B), (b12IgA2 [M+P]); (G) b12-IgG1; (H) b12-IgA2 monomer only (b12IgA2 [M]). Mice were challenged intravaginally with HIV-1 JR-CSF . (I) Average percent CD4 + T cells in CD3 T cells in PBMCs at each time points (n = 5-9). Statistical analysis was performed using unpaired t test. (* P = .0313 and *** P
Figure Legend Snippet: HIV-1 infection in humanized mice passively infused with b12-IgA or b12-IgG isotype. (A) Neutralization activity of anti-HIV human mAb b12-IgA. Reporter cell line TZM-bl cells that express CD4, CXCR4, CCR5, and a Tat-responsive reporter gene for luciferase were infected with 200 TCID 50 of replication-defective pseudovirus containing Env (SF162.LS) in the presence of various concentrations of anti-HIV monoclonal antibodies. Neutralization activity was measured by the reduction in luciferase reporter gene expression after a single round of pseudovirus infection in TZM-bl cells in triplicate. b12-IgG1 and the recombinant b12-IgA2 were compared at indicated concentrations. (B) Antibody level in circulation after passive transfer. NSG-hu mice were injected intravenously with various concentrations of purified b12-IgA2 (pIgA: mIgA = 1:1, mass ratio) or b12-IgG1 and the blood was collected after 4 hours. The plasma antibody concentrations were measured using ELISA. (n = 4-6, mean ± SEM). (C-D) Concentrations of b12 antibodies in plasma (C) or genital secretions (D) at the time of challenge. NSG-hu mice were injected intravenously with either 200 μg of b12-IgA2 or 20 μg of b12-IgG1 per mouse. Blood and genital secretions were collected after 4 hours. (E-I) Peripheral CD4 + T cell loss after HIV-1 challenge in NSG-hu mice injected with different b12 antibody isotypes; (E) purified human IgG/κ control antibody (hIgG/κ); (F) b12-IgA2 includes both monomeric and polymeric IgA as described in (B), (b12IgA2 [M+P]); (G) b12-IgG1; (H) b12-IgA2 monomer only (b12IgA2 [M]). Mice were challenged intravaginally with HIV-1 JR-CSF . (I) Average percent CD4 + T cells in CD3 T cells in PBMCs at each time points (n = 5-9). Statistical analysis was performed using unpaired t test. (* P = .0313 and *** P

Techniques Used: Infection, Mouse Assay, Neutralization, Activity Assay, Luciferase, Expressing, Recombinant, Injection, Purification, Enzyme-linked Immunosorbent Assay

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 "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

8) 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

9) Product Images from "GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome"

Article Title: GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006328

Knockdown of GPATCH3 enhances the activation of RLR-mediated signaling. (A) 293T cells (4 x 10 5 ) were cotransfected with Flag-GPATCH3, HA-actin (0.1 μg each) and control- or the indicated GPATCH3-shRNA plasmids (1 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblotting with anti-Flag or anti-HA antibodies (upper panel). 293T cells (4 x 10 5 ) were transfected with control- or the indicated GPATCH3-shRNA plasmids (1 μg each). Thirty-six hours after transfection, cell lysates were analyzed by immunoblotting with anti-GPATCH3 or anti-β-actin antibodies (lower panel). (B) 293T cells (1 x 10 5 ) were transfected with control- or the indicated GPATCH3-shRNA plasmids (0.25 μg each) together with the indicated reporters (0.05 μg each). Thirty-six hours later, cells were left uninfected or infected with SeV for 12 hours before luciferase assays were performed. (C) 293T cells (4 x 10 5 ) were transfected with control- or GPATCH3-shRNA plasmids (1 μg each). Thirty-six hours later, cells were left uninfected or infected with SeV for 12 hours before total RNAs were extracted and the mRNA levels of the indicated genes were analyzed by qPCR. (D) 293T cells (4 x 10 5 ) were transfected with control- or GPATCH3-shRNA plasmids (1 μg each). Thirty-six hours later, cells were left uninfected or infected with SeV for the indicated times. Cell lysates were then analyzed by immunoblotting with the indicated antibodies. (E F) A549 cells (4 x 10 5 ), HFFs (4 x 10 5 ) and PBMCs (1 x 10 6 ) were transfected with control or GPATCH3 siRNAs (40 nM). Thirty-six hours later, cells were left untreated or treated with the indicated infections or transfections. Twelve hours later, total RNAs were extracted and the mRNA levels of the indicated genes were analyzed by qPCR. Graphs show mean ± SD. n = 3. * P
Figure Legend Snippet: Knockdown of GPATCH3 enhances the activation of RLR-mediated signaling. (A) 293T cells (4 x 10 5 ) were cotransfected with Flag-GPATCH3, HA-actin (0.1 μg each) and control- or the indicated GPATCH3-shRNA plasmids (1 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblotting with anti-Flag or anti-HA antibodies (upper panel). 293T cells (4 x 10 5 ) were transfected with control- or the indicated GPATCH3-shRNA plasmids (1 μg each). Thirty-six hours after transfection, cell lysates were analyzed by immunoblotting with anti-GPATCH3 or anti-β-actin antibodies (lower panel). (B) 293T cells (1 x 10 5 ) were transfected with control- or the indicated GPATCH3-shRNA plasmids (0.25 μg each) together with the indicated reporters (0.05 μg each). Thirty-six hours later, cells were left uninfected or infected with SeV for 12 hours before luciferase assays were performed. (C) 293T cells (4 x 10 5 ) were transfected with control- or GPATCH3-shRNA plasmids (1 μg each). Thirty-six hours later, cells were left uninfected or infected with SeV for 12 hours before total RNAs were extracted and the mRNA levels of the indicated genes were analyzed by qPCR. (D) 293T cells (4 x 10 5 ) were transfected with control- or GPATCH3-shRNA plasmids (1 μg each). Thirty-six hours later, cells were left uninfected or infected with SeV for the indicated times. Cell lysates were then analyzed by immunoblotting with the indicated antibodies. (E F) A549 cells (4 x 10 5 ), HFFs (4 x 10 5 ) and PBMCs (1 x 10 6 ) were transfected with control or GPATCH3 siRNAs (40 nM). Thirty-six hours later, cells were left untreated or treated with the indicated infections or transfections. Twelve hours later, total RNAs were extracted and the mRNA levels of the indicated genes were analyzed by qPCR. Graphs show mean ± SD. n = 3. * P

Techniques Used: Activation Assay, shRNA, Transfection, Infection, Luciferase, Real-time Polymerase Chain Reaction

10) 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

11) 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

12) 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

13) 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

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Article Snippet: .. For passive antibody transfer experiments, NOD.Cg- Prkd scid IL2rg tm1Wjl /SzJ (NOD/SCID IL2r γ−/− , NSG) mice were transplanted intraperitoneally with 3 × 106 human PBMCs (AllCells) that were activated by PHA (5 μg/mL) for 4 days before transplantation (NSG-hu mice). .. Human cell engraftments in the mice were assessed at 3 to 5 weeks after transplantation.

Article Title: Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice
Article Snippet: .. NSG mice were transplanted intraperitoneally with 3 × 106 human PBMCs (AllCells) that were activated by PHA (5 μg/mL) for 4 days before transplantation (NSG-hu mice). .. Human cell engraftments in the mice were assessed at 3 to 5 weeks after transplantation.

Isolation:

Article Title: Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit
Article Snippet: .. Mixed lymphocyte reactions CD4+ T cells were isolated from PBMCs (AllCells, Alameda, California, USA) using EasySep human CD4+ T cells enrichment kit (StemCell Technologies, Vancouver, Canada). .. Dendritic cells (DCs) were generated by incubating PBMCs (Saily Bio, Shanghai, China) first with IL-4 (1000 U/ml) and GM-CSF (1000 U/ml), followed by maturation in media containing tumor necrosis factor (1000 U/ml), IL-1β (5 ng/ml), IL-6 (10 ng/ml), and prostaglandin E2 (1 μM) for 2 days.

Mouse Assay:

Article Title: Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice
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other:

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

Article Title: Potential Small Guide RNAs for tRNase ZL from Human Plasma, Peripheral Blood Mononuclear Cells, and Cultured Cell Lines
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Article Title: Inference and effects of barcode multiplets in droplet-based single-cell assays
Article Snippet: Profiling PBMCs using 10× scATAC-seq For 10× scATAC-seq experiments with PBMCs (PB003F, Allcells), frozen cells were quickly thawed in a 37 °C water bath for about 30 s and transferred to a 15 mL tube.

Purification:

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Article Snippet: .. PBMC used for NKT purification were obtained from AllCells, LLC (Alameda, CA) and were handled according to the manufacturer's direction. ..

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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.
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    <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.
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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

    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