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AllCells LLC human pbmcs
Detection of SMN Protein in SMNA Type 1 <t>PBMCs.</t> SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.
Human Pbmcs, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/human pbmcs/product/AllCells LLC
Average 92 stars, based on 3 article reviews
Price from $9.99 to $1999.99
human pbmcs - by Bioz Stars, 2020-09
92/100 stars

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1) Product Images from "Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses"

Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024269

Detection of SMN Protein in SMNA Type 1 PBMCs. SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.
Figure Legend Snippet: Detection of SMN Protein in SMNA Type 1 PBMCs. SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.

Techniques Used: Enzyme-linked Immunosorbent Assay

Quantitation of SMN by PBMC subtype. There was no significant difference in SMN protein levels between human lymphocytes and monocytes fractions separated from PBMCs. SMN protein was detectable in the majority of samples for both cell types, ranging from 6.8–25.8 pg/10 6 cells for lymphocytes and 8–17.5 pg/10 6 cells for monocytes. Error bars represent standard deviations.
Figure Legend Snippet: Quantitation of SMN by PBMC subtype. There was no significant difference in SMN protein levels between human lymphocytes and monocytes fractions separated from PBMCs. SMN protein was detectable in the majority of samples for both cell types, ranging from 6.8–25.8 pg/10 6 cells for lymphocytes and 8–17.5 pg/10 6 cells for monocytes. Error bars represent standard deviations.

Techniques Used: Quantitation Assay

ELISA Performance, parallelism and detection of SMN in human PBMCs. A : The recombinant hSMN standard curve data developed from N = 6 curves were highly reproducible with standard deviations of about ±0.23 OD unit variations. The dotted lines surrounding the dose-response curve represent 2 standard deviations. B : Comparison of SMN signal detection between recombinant human SMN (dilutions from 1∶4 to 1∶256) and SMN extracted from HeLa cell lysates (1∶4–1∶32) revealed a high degree of parallelism between reagents, allowing for accurate evaluation of native SMN protein using the recombinant SMN ELISA standard. C : The SMN ELISA detected protein in adult donor PBMCs; values ranged from 86 to 229 pg SMN per well at a dilution of 1∶4 and an average of 70.2 pg SMN protein per 10 6 cells. 10 7 PBMCs diluted 1∶4 through 1∶32 produced linear SMN protein levels. Even with only N = 6 normal samples, SMN levels in PBMCs varied by nearly 3-fold. Error bars represent standard deviations.
Figure Legend Snippet: ELISA Performance, parallelism and detection of SMN in human PBMCs. A : The recombinant hSMN standard curve data developed from N = 6 curves were highly reproducible with standard deviations of about ±0.23 OD unit variations. The dotted lines surrounding the dose-response curve represent 2 standard deviations. B : Comparison of SMN signal detection between recombinant human SMN (dilutions from 1∶4 to 1∶256) and SMN extracted from HeLa cell lysates (1∶4–1∶32) revealed a high degree of parallelism between reagents, allowing for accurate evaluation of native SMN protein using the recombinant SMN ELISA standard. C : The SMN ELISA detected protein in adult donor PBMCs; values ranged from 86 to 229 pg SMN per well at a dilution of 1∶4 and an average of 70.2 pg SMN protein per 10 6 cells. 10 7 PBMCs diluted 1∶4 through 1∶32 produced linear SMN protein levels. Even with only N = 6 normal samples, SMN levels in PBMCs varied by nearly 3-fold. Error bars represent standard deviations.

Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant, Produced

Smn protein levels by tissue in WT mice. Smn protein levels are distinct across the an array of tissues and regions of the brain. A : Smn levels varied by as much as 10-fold across tissue types in adult FVB mice (14 weeks old). SMN levels by tissue were distributed on a basis loosely ordered by having lesser to greater dividing cell populations. Smn levels for tissues are represented as Smn pg/mg total protein. PBMCs are represented as Smn pg/mg total protein in the lysis buffer extract. On a per cell basis the average Smn level was 67.2 pg/10 6 PBMCs. B : Smn protein in the brain, spinal cord, sciatic nerve and quadriceps muscle of wildtype mice shows marked variations, with spinal cord having 6-fold greater levels than muscle and nerve. In the brain, Smn protein signal in the thalamus was nearly 2.5-fold less than hippocampal levels. Error bars represent standard deviations.
Figure Legend Snippet: Smn protein levels by tissue in WT mice. Smn protein levels are distinct across the an array of tissues and regions of the brain. A : Smn levels varied by as much as 10-fold across tissue types in adult FVB mice (14 weeks old). SMN levels by tissue were distributed on a basis loosely ordered by having lesser to greater dividing cell populations. Smn levels for tissues are represented as Smn pg/mg total protein. PBMCs are represented as Smn pg/mg total protein in the lysis buffer extract. On a per cell basis the average Smn level was 67.2 pg/10 6 PBMCs. B : Smn protein in the brain, spinal cord, sciatic nerve and quadriceps muscle of wildtype mice shows marked variations, with spinal cord having 6-fold greater levels than muscle and nerve. In the brain, Smn protein signal in the thalamus was nearly 2.5-fold less than hippocampal levels. Error bars represent standard deviations.

Techniques Used: Mouse Assay, Lysis

2) Product Images from "Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFβ enhance the efficacy of cancer immunotherapy"

Article Title: Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFβ enhance the efficacy of cancer immunotherapy

Journal: Nature Communications

doi: 10.1038/s41467-017-02696-6

a -CTLA4-TGFβRII counteracts tumor-infiltrating Tregs and inhibits T H 17 differentiation. a Immunoblot analyses of the effect of a -CTLA4-TGFβRII or a -CTLA-4 on TGFβ-induced SMAD-2/3 phosphorylation and expression of FOXP3 in human PBMC. PBMCs were cultured for 24–48 h with rhIL-2 (100 IU ml −1 ) and anti-CD3/anti-CD28-coated Dynabeads in the presence or absence of rhTGFβ1 (2.5 ng ml −1 ) with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ). b , c Flow cytometric analysis of the in vivo effect of a -CTLA4-TGFβRII or a -CTLA-4 on bone marrow (BM) and tumor-infiltrating Tregs in human melanoma tumor-bearing NSG mice that were immune reconstituted with tumor-matched HLA A2 + human CD34 + bone marrow cells. Mice engrafted with human CD3 + cells were inoculated subcutaneously with either human melanoma tumor cells (A375 or SK-MEL-5) or patient-derived melanoma tumor xenografts (PDX-1 and PDX-2). Tumor-bearing mice (A375; SK-MEL-5; PDX-1; PDX-2) were randomized into groups and treated with either a -CTLA4-TGFβRII or a -CTLA-4 (5 mg kg −1 i.p. weekly), or vehicle alone (untreated control). b Representative flow data of FOXP3 expression in BM CD4 + CD25 + T cells in A375 or SK-MEL-5 tumor-bearing mice from each treatment or control group. c The percentage of Tregs (CD4 + /CD25 high /CD127 low /FOXP3 + ) in tumor-infiltrating lymphocytes in A375- or PDX-bearing mice (five animals per treatment group for A375 and four mice for PDXs). d Effect of a -CTLA4-TGFβRII or a -CTLA-4 on the suppression of tumor-reactive T cells by autologous Tregs in tumor-infiltrated BM from a patient. Marrow-infiltrating lymphocytes (MILs) stimulated with anti-CD3/anti-CD28-beads and rhIL-2 were carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) labeled and added to autologous BM that had been pulsed with either myeloma cell lysate (tumor-specific antigen) or nonspecific antigen in the presence or absence of autologous CD4 + /CD25 + MILs, with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ) for 3 days. Tumor antigen-reactive T cells (CD3 + /CFSE low /IFNγ + ) were quantified by flow cytometry (three in vitro replicates for each experimental group). e PBMCs were stimulated with anti-CD3/CD28 beads in the presence of IL17-skewing cytokines (10 ng ml −1 IL-6, 5 ng ml −1 TGFβ, 10 ng ml −1 anti–IFN-γ, and 10 ng ml −1 anti–IL-4) with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ) for 3 days. Following addition of Leukocyte Activation Cocktail (2 μl ml −1 for 4 h), cells were analyzed by flow cytometry for intracellular expression of IL-17 and IFN-γ in CD4 + T cells (three in vitro replicates for each experimental group). Asterisks represent statistical significance between the a -CTLA4-TGFβRII and a -CTLA-4 treatments. Representative flow cytometric dot plots are shown
Figure Legend Snippet: a -CTLA4-TGFβRII counteracts tumor-infiltrating Tregs and inhibits T H 17 differentiation. a Immunoblot analyses of the effect of a -CTLA4-TGFβRII or a -CTLA-4 on TGFβ-induced SMAD-2/3 phosphorylation and expression of FOXP3 in human PBMC. PBMCs were cultured for 24–48 h with rhIL-2 (100 IU ml −1 ) and anti-CD3/anti-CD28-coated Dynabeads in the presence or absence of rhTGFβ1 (2.5 ng ml −1 ) with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ). b , c Flow cytometric analysis of the in vivo effect of a -CTLA4-TGFβRII or a -CTLA-4 on bone marrow (BM) and tumor-infiltrating Tregs in human melanoma tumor-bearing NSG mice that were immune reconstituted with tumor-matched HLA A2 + human CD34 + bone marrow cells. Mice engrafted with human CD3 + cells were inoculated subcutaneously with either human melanoma tumor cells (A375 or SK-MEL-5) or patient-derived melanoma tumor xenografts (PDX-1 and PDX-2). Tumor-bearing mice (A375; SK-MEL-5; PDX-1; PDX-2) were randomized into groups and treated with either a -CTLA4-TGFβRII or a -CTLA-4 (5 mg kg −1 i.p. weekly), or vehicle alone (untreated control). b Representative flow data of FOXP3 expression in BM CD4 + CD25 + T cells in A375 or SK-MEL-5 tumor-bearing mice from each treatment or control group. c The percentage of Tregs (CD4 + /CD25 high /CD127 low /FOXP3 + ) in tumor-infiltrating lymphocytes in A375- or PDX-bearing mice (five animals per treatment group for A375 and four mice for PDXs). d Effect of a -CTLA4-TGFβRII or a -CTLA-4 on the suppression of tumor-reactive T cells by autologous Tregs in tumor-infiltrated BM from a patient. Marrow-infiltrating lymphocytes (MILs) stimulated with anti-CD3/anti-CD28-beads and rhIL-2 were carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) labeled and added to autologous BM that had been pulsed with either myeloma cell lysate (tumor-specific antigen) or nonspecific antigen in the presence or absence of autologous CD4 + /CD25 + MILs, with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ) for 3 days. Tumor antigen-reactive T cells (CD3 + /CFSE low /IFNγ + ) were quantified by flow cytometry (three in vitro replicates for each experimental group). e PBMCs were stimulated with anti-CD3/CD28 beads in the presence of IL17-skewing cytokines (10 ng ml −1 IL-6, 5 ng ml −1 TGFβ, 10 ng ml −1 anti–IFN-γ, and 10 ng ml −1 anti–IL-4) with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ) for 3 days. Following addition of Leukocyte Activation Cocktail (2 μl ml −1 for 4 h), cells were analyzed by flow cytometry for intracellular expression of IL-17 and IFN-γ in CD4 + T cells (three in vitro replicates for each experimental group). Asterisks represent statistical significance between the a -CTLA4-TGFβRII and a -CTLA-4 treatments. Representative flow cytometric dot plots are shown

Techniques Used: Expressing, Cell Culture, Flow Cytometry, In Vivo, Mouse Assay, Derivative Assay, Labeling, Cytometry, In Vitro, Activation Assay

3) Product Images from "Mesenchymal Stem Cells Derived from Human Gingiva Are Capable of Immunomodulatory Functions and Ameliorate Inflammation-Related Tissue Destruction in Experimental Colitis 1"

Article Title: Mesenchymal Stem Cells Derived from Human Gingiva Are Capable of Immunomodulatory Functions and Ameliorate Inflammation-Related Tissue Destruction in Experimental Colitis 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.0902318

IFN- γ -induced IDO expression and IL-10 secretion by GMSCs. A–C , GMSCs or BMSCs were stimulated with increasing concentrations of IFN- γ for 24 h. Then the expression of IDO protein was determined by Western blot, while the IDO activity was analyzed by measuring the concentration of kynurenine in the conditioned medium ( A ). IFN- γ -induced IL-10 secretion in the supernatants was determined by using ELISA ( B ), whereas the expression of iNOS and COX-2 in MSCs in response to IFN- γ was determined by Western blot ( C ). D–F , Two × 10 5 PBMCs were cultured alone or cocultured with the same number of GMSCs or BMSCs under cell-cell contact conditions in the presence or absence of 5 μ g/ml PHA for 72 h. Afterward, the concentrations of IFN- γ ( D ) and IL-10 ( E ) in the supernatants were determined by using ELISA, whereas IDO protein expression and activity were determined by Western blot and kynurenine assay, respectively ( F ). G and H , PBMCs were pretreated for 2 h with different concentrations of specific neutralizing Ab for human IFN- γ (0.5–10 μ g/ml) or an isotype-matched mouse IgG (10 μ g/ml), followed by coculturing with the same number of GMSCs (1/1) under cell-cell contact conditions in the presence or absence of 5 μ g/ml PHA for 72 h. Then IDO protein expression and activity were determined by Western blot and kynurenine assay, respectively ( G ), whereas the concentration of IL-10 in the supernatants was determined by using ELISA ( H ). *, p
Figure Legend Snippet: IFN- γ -induced IDO expression and IL-10 secretion by GMSCs. A–C , GMSCs or BMSCs were stimulated with increasing concentrations of IFN- γ for 24 h. Then the expression of IDO protein was determined by Western blot, while the IDO activity was analyzed by measuring the concentration of kynurenine in the conditioned medium ( A ). IFN- γ -induced IL-10 secretion in the supernatants was determined by using ELISA ( B ), whereas the expression of iNOS and COX-2 in MSCs in response to IFN- γ was determined by Western blot ( C ). D–F , Two × 10 5 PBMCs were cultured alone or cocultured with the same number of GMSCs or BMSCs under cell-cell contact conditions in the presence or absence of 5 μ g/ml PHA for 72 h. Afterward, the concentrations of IFN- γ ( D ) and IL-10 ( E ) in the supernatants were determined by using ELISA, whereas IDO protein expression and activity were determined by Western blot and kynurenine assay, respectively ( F ). G and H , PBMCs were pretreated for 2 h with different concentrations of specific neutralizing Ab for human IFN- γ (0.5–10 μ g/ml) or an isotype-matched mouse IgG (10 μ g/ml), followed by coculturing with the same number of GMSCs (1/1) under cell-cell contact conditions in the presence or absence of 5 μ g/ml PHA for 72 h. Then IDO protein expression and activity were determined by Western blot and kynurenine assay, respectively ( G ), whereas the concentration of IL-10 in the supernatants was determined by using ELISA ( H ). *, p

Techniques Used: Expressing, Western Blot, Activity Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Related Articles

Cell Isolation:

Article Title: Discovery and preclinical characterization of the antagonist anti-PD-L1 monoclonal antibody LY3300054
Article Snippet: .. CD4+ T cells were purified from fresh human PBMC of a different healthy donor (AllCells) using Human CD4+ T Cell Isolation Kit (Miltenyi). ..

Centrifugation:

Article Title: Targeting the Ion Channel Kv1.3 with Scorpion Venom Peptides Engineered for Potency, Selectivity, and Half-life
Article Snippet: .. Human PBMCs were obtained from AllCells LLC or purified from healthy donor blood by Ficoll-Paque (GE Healthcare) density centrifugation. .. Purified PBMCs were plated in 96-well culture plates at 1 × 106 cells/well in RPMI medium supplemented with 10% FBS and incubated overnight with Kv1.3 blockers.

Cell Culture:

Article Title: Evidence Suggesting a Role of Iron in a Mouse Model of Nephrogenic Systemic Fibrosis
Article Snippet: .. Cell culture studies We have recently shown that Omniscan induces the differentiation of human PBMC into ferroportin-expressing, fibrocyte-like, spindle-shaped cells [ ]. ..

Purification:

Article Title: Discovery and preclinical characterization of the antagonist anti-PD-L1 monoclonal antibody LY3300054
Article Snippet: .. CD4+ T cells were purified from fresh human PBMC of a different healthy donor (AllCells) using Human CD4+ T Cell Isolation Kit (Miltenyi). ..

Article Title: Targeting the Ion Channel Kv1.3 with Scorpion Venom Peptides Engineered for Potency, Selectivity, and Half-life
Article Snippet: .. Human PBMCs were obtained from AllCells LLC or purified from healthy donor blood by Ficoll-Paque (GE Healthcare) density centrifugation. .. Purified PBMCs were plated in 96-well culture plates at 1 × 106 cells/well in RPMI medium supplemented with 10% FBS and incubated overnight with Kv1.3 blockers.

other:

Article Title: Closing two doors of viral entry: Intramolecular combination of a coreceptor- and fusion inhibitor of HIV-1
Article Snippet: Human embryonic kidney cells 293-T and HEK293-EBNA cells were obtained from ATCC, Manassas, VA. Human PBMC were obtained from AllCells (Emeryville, CA), stimulated for one day in PBMC media (RPMI-1640 media containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium-pyruvate, 0.1 mM MEM non-essential amino acids) supplemented with 2 μg/mL phytohemagglutinin (all Invitrogen) and maintained in PBMC media containing 5 units/mL human IL-2 (Roche Applied Science, Indianapolis, IN).

Article Title: Mesenchymal Stem Cells Derived from Human Gingiva Are Capable of Immunomodulatory Functions and Ameliorate Inflammation-Related Tissue Destruction in Experimental Colitis 1
Article Snippet: To this end, GMSCs or BMSCs were cocultured under cell-cell contact or Transwell systems with increasing numbers of human PBMCs in the presence of PHA for 72 h. Our results showed that GMSCs, similar to BMSCs, inhibited mitogen-stimulated PBMC proliferation in a cell density-dependent manner under both cell-cell contact and Transwell cultures ( ).

Staining:

Article Title: Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFβ enhance the efficacy of cancer immunotherapy
Article Snippet: .. Human PBMCs (ALLCELLS) were stained with anti-CD3 and Glycophorin A to enumerate T cells. .. Cells were cultured with rhIL-2 (100 IU ml−1 ) and anti-CD3/anti-CD28-coated Dynabeads (Life Technologies) at a ratio of 1 : 3 (cell : bead) in the presence or absence of 2.5 ng ml−1 rhTGF-β1 with or without either a -CTLA4-TGFβRII or a -CTLA-4 antibody (5 μg ml−1 ).

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    AllCells LLC adcc assay human peripheral blood mononuclear cells pbmc
    <t>ADCC</t> of Y-443 and Fc-mutated antibodies against MDA-MB-231 cells. MDA-MB-231 cells pre-labeled with Calcein AM were incubated with different concentrations of Y-443 or the Fc mutants, followed by addition of <t>PBMC</t> effector cells at a ratio of 1:50. The cell mixture was incubated for 4 hours at 37°C, and Calcein AM intensity in cells was detected by Acumen eX3 (TTP labtech). The results are the mean ± S.D. of dead cell ratio.
    Adcc Assay Human Peripheral Blood Mononuclear Cells Pbmc, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adcc assay human peripheral blood mononuclear cells pbmc/product/AllCells LLC
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    adcc assay human peripheral blood mononuclear cells pbmc - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    92
    AllCells LLC human pbmcs
    Detection of SMN Protein in SMNA Type 1 <t>PBMCs.</t> SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.
    Human Pbmcs, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human pbmcs/product/AllCells LLC
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    human pbmcs - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

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    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

    Detection of SMN Protein in SMNA Type 1 PBMCs. SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: Detection of SMN Protein in SMNA Type 1 PBMCs. SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Enzyme-linked Immunosorbent Assay

    Quantitation of SMN by PBMC subtype. There was no significant difference in SMN protein levels between human lymphocytes and monocytes fractions separated from PBMCs. SMN protein was detectable in the majority of samples for both cell types, ranging from 6.8–25.8 pg/10 6 cells for lymphocytes and 8–17.5 pg/10 6 cells for monocytes. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: Quantitation of SMN by PBMC subtype. There was no significant difference in SMN protein levels between human lymphocytes and monocytes fractions separated from PBMCs. SMN protein was detectable in the majority of samples for both cell types, ranging from 6.8–25.8 pg/10 6 cells for lymphocytes and 8–17.5 pg/10 6 cells for monocytes. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Quantitation Assay

    ELISA Performance, parallelism and detection of SMN in human PBMCs. A : The recombinant hSMN standard curve data developed from N = 6 curves were highly reproducible with standard deviations of about ±0.23 OD unit variations. The dotted lines surrounding the dose-response curve represent 2 standard deviations. B : Comparison of SMN signal detection between recombinant human SMN (dilutions from 1∶4 to 1∶256) and SMN extracted from HeLa cell lysates (1∶4–1∶32) revealed a high degree of parallelism between reagents, allowing for accurate evaluation of native SMN protein using the recombinant SMN ELISA standard. C : The SMN ELISA detected protein in adult donor PBMCs; values ranged from 86 to 229 pg SMN per well at a dilution of 1∶4 and an average of 70.2 pg SMN protein per 10 6 cells. 10 7 PBMCs diluted 1∶4 through 1∶32 produced linear SMN protein levels. Even with only N = 6 normal samples, SMN levels in PBMCs varied by nearly 3-fold. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: ELISA Performance, parallelism and detection of SMN in human PBMCs. A : The recombinant hSMN standard curve data developed from N = 6 curves were highly reproducible with standard deviations of about ±0.23 OD unit variations. The dotted lines surrounding the dose-response curve represent 2 standard deviations. B : Comparison of SMN signal detection between recombinant human SMN (dilutions from 1∶4 to 1∶256) and SMN extracted from HeLa cell lysates (1∶4–1∶32) revealed a high degree of parallelism between reagents, allowing for accurate evaluation of native SMN protein using the recombinant SMN ELISA standard. C : The SMN ELISA detected protein in adult donor PBMCs; values ranged from 86 to 229 pg SMN per well at a dilution of 1∶4 and an average of 70.2 pg SMN protein per 10 6 cells. 10 7 PBMCs diluted 1∶4 through 1∶32 produced linear SMN protein levels. Even with only N = 6 normal samples, SMN levels in PBMCs varied by nearly 3-fold. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Produced

    Smn protein levels by tissue in WT mice. Smn protein levels are distinct across the an array of tissues and regions of the brain. A : Smn levels varied by as much as 10-fold across tissue types in adult FVB mice (14 weeks old). SMN levels by tissue were distributed on a basis loosely ordered by having lesser to greater dividing cell populations. Smn levels for tissues are represented as Smn pg/mg total protein. PBMCs are represented as Smn pg/mg total protein in the lysis buffer extract. On a per cell basis the average Smn level was 67.2 pg/10 6 PBMCs. B : Smn protein in the brain, spinal cord, sciatic nerve and quadriceps muscle of wildtype mice shows marked variations, with spinal cord having 6-fold greater levels than muscle and nerve. In the brain, Smn protein signal in the thalamus was nearly 2.5-fold less than hippocampal levels. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: Smn protein levels by tissue in WT mice. Smn protein levels are distinct across the an array of tissues and regions of the brain. A : Smn levels varied by as much as 10-fold across tissue types in adult FVB mice (14 weeks old). SMN levels by tissue were distributed on a basis loosely ordered by having lesser to greater dividing cell populations. Smn levels for tissues are represented as Smn pg/mg total protein. PBMCs are represented as Smn pg/mg total protein in the lysis buffer extract. On a per cell basis the average Smn level was 67.2 pg/10 6 PBMCs. B : Smn protein in the brain, spinal cord, sciatic nerve and quadriceps muscle of wildtype mice shows marked variations, with spinal cord having 6-fold greater levels than muscle and nerve. In the brain, Smn protein signal in the thalamus was nearly 2.5-fold less than hippocampal levels. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Mouse Assay, Lysis

    a -CTLA4-TGFβRII counteracts tumor-infiltrating Tregs and inhibits T H 17 differentiation. a Immunoblot analyses of the effect of a -CTLA4-TGFβRII or a -CTLA-4 on TGFβ-induced SMAD-2/3 phosphorylation and expression of FOXP3 in human PBMC. PBMCs were cultured for 24–48 h with rhIL-2 (100 IU ml −1 ) and anti-CD3/anti-CD28-coated Dynabeads in the presence or absence of rhTGFβ1 (2.5 ng ml −1 ) with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ). b , c Flow cytometric analysis of the in vivo effect of a -CTLA4-TGFβRII or a -CTLA-4 on bone marrow (BM) and tumor-infiltrating Tregs in human melanoma tumor-bearing NSG mice that were immune reconstituted with tumor-matched HLA A2 + human CD34 + bone marrow cells. Mice engrafted with human CD3 + cells were inoculated subcutaneously with either human melanoma tumor cells (A375 or SK-MEL-5) or patient-derived melanoma tumor xenografts (PDX-1 and PDX-2). Tumor-bearing mice (A375; SK-MEL-5; PDX-1; PDX-2) were randomized into groups and treated with either a -CTLA4-TGFβRII or a -CTLA-4 (5 mg kg −1 i.p. weekly), or vehicle alone (untreated control). b Representative flow data of FOXP3 expression in BM CD4 + CD25 + T cells in A375 or SK-MEL-5 tumor-bearing mice from each treatment or control group. c The percentage of Tregs (CD4 + /CD25 high /CD127 low /FOXP3 + ) in tumor-infiltrating lymphocytes in A375- or PDX-bearing mice (five animals per treatment group for A375 and four mice for PDXs). d Effect of a -CTLA4-TGFβRII or a -CTLA-4 on the suppression of tumor-reactive T cells by autologous Tregs in tumor-infiltrated BM from a patient. Marrow-infiltrating lymphocytes (MILs) stimulated with anti-CD3/anti-CD28-beads and rhIL-2 were carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) labeled and added to autologous BM that had been pulsed with either myeloma cell lysate (tumor-specific antigen) or nonspecific antigen in the presence or absence of autologous CD4 + /CD25 + MILs, with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ) for 3 days. Tumor antigen-reactive T cells (CD3 + /CFSE low /IFNγ + ) were quantified by flow cytometry (three in vitro replicates for each experimental group). e PBMCs were stimulated with anti-CD3/CD28 beads in the presence of IL17-skewing cytokines (10 ng ml −1 IL-6, 5 ng ml −1 TGFβ, 10 ng ml −1 anti–IFN-γ, and 10 ng ml −1 anti–IL-4) with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ) for 3 days. Following addition of Leukocyte Activation Cocktail (2 μl ml −1 for 4 h), cells were analyzed by flow cytometry for intracellular expression of IL-17 and IFN-γ in CD4 + T cells (three in vitro replicates for each experimental group). Asterisks represent statistical significance between the a -CTLA4-TGFβRII and a -CTLA-4 treatments. Representative flow cytometric dot plots are shown

    Journal: Nature Communications

    Article Title: Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFβ enhance the efficacy of cancer immunotherapy

    doi: 10.1038/s41467-017-02696-6

    Figure Lengend Snippet: a -CTLA4-TGFβRII counteracts tumor-infiltrating Tregs and inhibits T H 17 differentiation. a Immunoblot analyses of the effect of a -CTLA4-TGFβRII or a -CTLA-4 on TGFβ-induced SMAD-2/3 phosphorylation and expression of FOXP3 in human PBMC. PBMCs were cultured for 24–48 h with rhIL-2 (100 IU ml −1 ) and anti-CD3/anti-CD28-coated Dynabeads in the presence or absence of rhTGFβ1 (2.5 ng ml −1 ) with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ). b , c Flow cytometric analysis of the in vivo effect of a -CTLA4-TGFβRII or a -CTLA-4 on bone marrow (BM) and tumor-infiltrating Tregs in human melanoma tumor-bearing NSG mice that were immune reconstituted with tumor-matched HLA A2 + human CD34 + bone marrow cells. Mice engrafted with human CD3 + cells were inoculated subcutaneously with either human melanoma tumor cells (A375 or SK-MEL-5) or patient-derived melanoma tumor xenografts (PDX-1 and PDX-2). Tumor-bearing mice (A375; SK-MEL-5; PDX-1; PDX-2) were randomized into groups and treated with either a -CTLA4-TGFβRII or a -CTLA-4 (5 mg kg −1 i.p. weekly), or vehicle alone (untreated control). b Representative flow data of FOXP3 expression in BM CD4 + CD25 + T cells in A375 or SK-MEL-5 tumor-bearing mice from each treatment or control group. c The percentage of Tregs (CD4 + /CD25 high /CD127 low /FOXP3 + ) in tumor-infiltrating lymphocytes in A375- or PDX-bearing mice (five animals per treatment group for A375 and four mice for PDXs). d Effect of a -CTLA4-TGFβRII or a -CTLA-4 on the suppression of tumor-reactive T cells by autologous Tregs in tumor-infiltrated BM from a patient. Marrow-infiltrating lymphocytes (MILs) stimulated with anti-CD3/anti-CD28-beads and rhIL-2 were carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) labeled and added to autologous BM that had been pulsed with either myeloma cell lysate (tumor-specific antigen) or nonspecific antigen in the presence or absence of autologous CD4 + /CD25 + MILs, with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ) for 3 days. Tumor antigen-reactive T cells (CD3 + /CFSE low /IFNγ + ) were quantified by flow cytometry (three in vitro replicates for each experimental group). e PBMCs were stimulated with anti-CD3/CD28 beads in the presence of IL17-skewing cytokines (10 ng ml −1 IL-6, 5 ng ml −1 TGFβ, 10 ng ml −1 anti–IFN-γ, and 10 ng ml −1 anti–IL-4) with or without either a -CTLA4-TGFβRII or a -CTLA-4 (5 μg ml −1 ) for 3 days. Following addition of Leukocyte Activation Cocktail (2 μl ml −1 for 4 h), cells were analyzed by flow cytometry for intracellular expression of IL-17 and IFN-γ in CD4 + T cells (three in vitro replicates for each experimental group). Asterisks represent statistical significance between the a -CTLA4-TGFβRII and a -CTLA-4 treatments. Representative flow cytometric dot plots are shown

    Article Snippet: Human PBMCs (ALLCELLS) were stained with anti-CD3 and Glycophorin A to enumerate T cells.

    Techniques: Expressing, Cell Culture, Flow Cytometry, In Vivo, Mouse Assay, Derivative Assay, Labeling, Cytometry, In Vitro, Activation Assay

    Combination of LY3300054 and ipilimumab enhances T cell activation in vitro. Panel a : Mixed leukocyte reactions. Allogeneic DC were co-cultured with purified CD4 + T cells for 72 h in the presence of increasing (two-fold increments) concentrations of LY3300054, ipilimumab or a combination of both antibodies ranging from 0.0003 to 67 nM. Supernatants were measured for IFN-γ and IL-2 production by ELISA. Each data point represents the average of 8 replicates, with error bars representing the SEM. Data were generated with four different PBMC donors. Panel b : Gene expression analysis of cell lysate from the mixed leukocyte reactions was performed using QuantiGene Plex assay. Venn diagram showing the number of shared (overlap circle) and treatment-specific (no overlap) DEGs across the different treatments. Tables list the Log2 fold-change of LY3300054 vs control group for genes with fold-change > 1.5, p value

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Discovery and preclinical characterization of the antagonist anti-PD-L1 monoclonal antibody LY3300054

    doi: 10.1186/s40425-018-0329-7

    Figure Lengend Snippet: Combination of LY3300054 and ipilimumab enhances T cell activation in vitro. Panel a : Mixed leukocyte reactions. Allogeneic DC were co-cultured with purified CD4 + T cells for 72 h in the presence of increasing (two-fold increments) concentrations of LY3300054, ipilimumab or a combination of both antibodies ranging from 0.0003 to 67 nM. Supernatants were measured for IFN-γ and IL-2 production by ELISA. Each data point represents the average of 8 replicates, with error bars representing the SEM. Data were generated with four different PBMC donors. Panel b : Gene expression analysis of cell lysate from the mixed leukocyte reactions was performed using QuantiGene Plex assay. Venn diagram showing the number of shared (overlap circle) and treatment-specific (no overlap) DEGs across the different treatments. Tables list the Log2 fold-change of LY3300054 vs control group for genes with fold-change > 1.5, p value

    Article Snippet: CD4+ T cells were purified from fresh human PBMC of a different healthy donor (AllCells) using Human CD4+ T Cell Isolation Kit (Miltenyi).

    Techniques: Activation Assay, In Vitro, Cell Culture, Purification, Enzyme-linked Immunosorbent Assay, Generated, Expressing, Plex Assay