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Corning Life Sciences pbmc
R10015 inhibits R5 and X4 <t>HIV-1</t> latent infection of resting CD4 T cells and primary isolate infection of <t>PBMC.</t> (A) R10015 inhibits HIV latent infection of resting CD4 + T cells. Cells were treated with R10015 (100 μM) or DMSO for 1 h and infected with HIV-1(NL4-3) for 2 h. The virus and the drug were washed away, and the cells were cultured for 5 days in the absence of R10015 and then activated with CD3/CD28 beads. Viral p24 release was measured. (B) CD25 and CD69 surface staining demonstrates that R10015 did not inhibit T cell activation with this short period of drug treatment. (C) R10015 inhibits R5 HIV-1 latent infection of CD45RO + memory CD4 T cells. Resting memory CD4 T cells were similarly treated with R10015, infected with HIV-1(AD8), washed, incubated for 5 days without stimulation, and then activated with CD3/CD28 beads. (D) CD69 surface staining was performed for control of R10015 effects on T cell activation. (E) R10015 inhibits HIV-1 primary isolate infection of PBMC. PBMC were cultured for 1 day and then treated with 100 μM R10015 for 1 h. The cells were infected with HIV 92/BR/018 (Brazil) or HIV 93UG070 (Uganda) for 3 h, washed to remove the viruses and R10015, and cultured for 3 days. The supernatant was analyzed for HIV-1 p24 by ELISA. DMSO was used as a control. The error bars indicate standard deviations.
Pbmc, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pbmc - by Bioz Stars, 2021-03
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1) Product Images from "Discovery of Novel Small-Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1"

Article Title: Discovery of Novel Small-Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1

Journal: Journal of Virology

doi: 10.1128/JVI.02418-16

R10015 inhibits R5 and X4 HIV-1 latent infection of resting CD4 T cells and primary isolate infection of PBMC. (A) R10015 inhibits HIV latent infection of resting CD4 + T cells. Cells were treated with R10015 (100 μM) or DMSO for 1 h and infected with HIV-1(NL4-3) for 2 h. The virus and the drug were washed away, and the cells were cultured for 5 days in the absence of R10015 and then activated with CD3/CD28 beads. Viral p24 release was measured. (B) CD25 and CD69 surface staining demonstrates that R10015 did not inhibit T cell activation with this short period of drug treatment. (C) R10015 inhibits R5 HIV-1 latent infection of CD45RO + memory CD4 T cells. Resting memory CD4 T cells were similarly treated with R10015, infected with HIV-1(AD8), washed, incubated for 5 days without stimulation, and then activated with CD3/CD28 beads. (D) CD69 surface staining was performed for control of R10015 effects on T cell activation. (E) R10015 inhibits HIV-1 primary isolate infection of PBMC. PBMC were cultured for 1 day and then treated with 100 μM R10015 for 1 h. The cells were infected with HIV 92/BR/018 (Brazil) or HIV 93UG070 (Uganda) for 3 h, washed to remove the viruses and R10015, and cultured for 3 days. The supernatant was analyzed for HIV-1 p24 by ELISA. DMSO was used as a control. The error bars indicate standard deviations.
Figure Legend Snippet: R10015 inhibits R5 and X4 HIV-1 latent infection of resting CD4 T cells and primary isolate infection of PBMC. (A) R10015 inhibits HIV latent infection of resting CD4 + T cells. Cells were treated with R10015 (100 μM) or DMSO for 1 h and infected with HIV-1(NL4-3) for 2 h. The virus and the drug were washed away, and the cells were cultured for 5 days in the absence of R10015 and then activated with CD3/CD28 beads. Viral p24 release was measured. (B) CD25 and CD69 surface staining demonstrates that R10015 did not inhibit T cell activation with this short period of drug treatment. (C) R10015 inhibits R5 HIV-1 latent infection of CD45RO + memory CD4 T cells. Resting memory CD4 T cells were similarly treated with R10015, infected with HIV-1(AD8), washed, incubated for 5 days without stimulation, and then activated with CD3/CD28 beads. (D) CD69 surface staining was performed for control of R10015 effects on T cell activation. (E) R10015 inhibits HIV-1 primary isolate infection of PBMC. PBMC were cultured for 1 day and then treated with 100 μM R10015 for 1 h. The cells were infected with HIV 92/BR/018 (Brazil) or HIV 93UG070 (Uganda) for 3 h, washed to remove the viruses and R10015, and cultured for 3 days. The supernatant was analyzed for HIV-1 p24 by ELISA. DMSO was used as a control. The error bars indicate standard deviations.

Techniques Used: Infection, Cell Culture, Staining, Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

2) Product Images from "Differential CD4 T Regulatory Cell Phenotype Induced by Andes Hantavirus Glycoprotein"

Article Title: Differential CD4 T Regulatory Cell Phenotype Induced by Andes Hantavirus Glycoprotein

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2020.00430

ANDV-GP modulates CD4 + Treg phenotype by down regulating CXCR3. Three healthy donors were used to isolate CD4 + Treg from PBMCs, stimulated with VLP or no-treated (mock) and analyzed after 3 days in cell culture. (A) Mean fluorescence intensity (MFI) of T-bet, GATA3, and RORγt that allows the detection of Th1-like Treg, Th2-like Treg, and Th17-like Treg, respectively. (B) Frequency and expression measured by MFI of CXCR3 on the T-bet + CD4 + Treg population. Each line represents a subject sample ( n = 3). * P
Figure Legend Snippet: ANDV-GP modulates CD4 + Treg phenotype by down regulating CXCR3. Three healthy donors were used to isolate CD4 + Treg from PBMCs, stimulated with VLP or no-treated (mock) and analyzed after 3 days in cell culture. (A) Mean fluorescence intensity (MFI) of T-bet, GATA3, and RORγt that allows the detection of Th1-like Treg, Th2-like Treg, and Th17-like Treg, respectively. (B) Frequency and expression measured by MFI of CXCR3 on the T-bet + CD4 + Treg population. Each line represents a subject sample ( n = 3). * P

Techniques Used: Cell Culture, Fluorescence, Expressing

Increase frequency of memory CD4 + Treg PD-1 + on HCPS survivors. (A) Gating strategy of a representative sample showing CD4 + , CD4 + CD45RO + , CD127 low/− CD25 + , CD127 low/− CD25 + FoxP3 + , memory Treg PD1 + , and memory Treg CTLA-4 + cells. (B) Frequency of total CD4 + T cells gated in live PBMC. (C) Frequency of memory CD4 + T cells CD4 + CD45RO + . (D) Frequency of memory CD4 Treg (CD4 + CD45RO + CD25 + CD127 low/− FoxP3 + ). (E) Frequency of memory Treg PD-1 + (left) and memory Treg CTLA-4 + (right). Data represented as dots (HD n = 15) and squares (HCPS n = 16), indicating the mean ± SD. ** P = 0.0021 by unpaired Student's t -test.
Figure Legend Snippet: Increase frequency of memory CD4 + Treg PD-1 + on HCPS survivors. (A) Gating strategy of a representative sample showing CD4 + , CD4 + CD45RO + , CD127 low/− CD25 + , CD127 low/− CD25 + FoxP3 + , memory Treg PD1 + , and memory Treg CTLA-4 + cells. (B) Frequency of total CD4 + T cells gated in live PBMC. (C) Frequency of memory CD4 + T cells CD4 + CD45RO + . (D) Frequency of memory CD4 Treg (CD4 + CD45RO + CD25 + CD127 low/− FoxP3 + ). (E) Frequency of memory Treg PD-1 + (left) and memory Treg CTLA-4 + (right). Data represented as dots (HD n = 15) and squares (HCPS n = 16), indicating the mean ± SD. ** P = 0.0021 by unpaired Student's t -test.

Techniques Used:

ANDV-GP virus like particles induces a Th2-like phenotype on memory CD4 + Treg cells. (A) ANDV-GP VLPs are detected in CD4 + T cells. PBMC were incubated with VLPs and detected by flow cytometry using an anti-ANDV-GP Qdot655 conjugated antibody (left). Purified CD4 + T cells were incubated with VLP and ANDV-GP was detected with anti-ANDV-GP Qdot655 conjugated antibody and visualized in CD4 + CXCR3 + cells by confocal microscopy, arrows indicate the intracellular ANDV-GP staining (right). Following the same gating strategy of Figure 2 was evaluated the effect of ANDV-GP VLP on Th-like Treg frequency. (B) Frequency of total Treg (CD4 + CD127 low/− CD25 + ) in HCPS and HD PBMCs after VLP overnight stimulation follow by analysis at day 7 of cell culture. (C) Frequency of memory CD4 + Treg CD45RA − CCR4 + in HCPS and HD in mock and VLP stimulus. (D) Frequency of Th-like memory CD4 + Treg CCR4 + in HCPS (above panel) and HD (below panel) in mock and VLP conditions. Data for each individual is represented as a line connecting mock and VLP conditions (HD n = 7; HCPS n = 7), * P
Figure Legend Snippet: ANDV-GP virus like particles induces a Th2-like phenotype on memory CD4 + Treg cells. (A) ANDV-GP VLPs are detected in CD4 + T cells. PBMC were incubated with VLPs and detected by flow cytometry using an anti-ANDV-GP Qdot655 conjugated antibody (left). Purified CD4 + T cells were incubated with VLP and ANDV-GP was detected with anti-ANDV-GP Qdot655 conjugated antibody and visualized in CD4 + CXCR3 + cells by confocal microscopy, arrows indicate the intracellular ANDV-GP staining (right). Following the same gating strategy of Figure 2 was evaluated the effect of ANDV-GP VLP on Th-like Treg frequency. (B) Frequency of total Treg (CD4 + CD127 low/− CD25 + ) in HCPS and HD PBMCs after VLP overnight stimulation follow by analysis at day 7 of cell culture. (C) Frequency of memory CD4 + Treg CD45RA − CCR4 + in HCPS and HD in mock and VLP stimulus. (D) Frequency of Th-like memory CD4 + Treg CCR4 + in HCPS (above panel) and HD (below panel) in mock and VLP conditions. Data for each individual is represented as a line connecting mock and VLP conditions (HD n = 7; HCPS n = 7), * P

Techniques Used: Incubation, Flow Cytometry, Purification, Confocal Microscopy, Staining, Cell Culture

3) Product Images from "Anti-Tumor Activity and Immunotherapeutic Potential of a Bisphosphonate Prodrug"

Article Title: Anti-Tumor Activity and Immunotherapeutic Potential of a Bisphosphonate Prodrug

Journal: Scientific Reports

doi: 10.1038/s41598-017-05553-0

Selective activation of Vγ2Vδ2 T cells by compound 7 . ( A ) Selective expansion of Vγ2Vδ2 T cells from blood αβ and γδ T cells after culture with compound 7 . PBMC from a prostate cancer patient were stimulated with 1 μM compound 7 and IL-2. The two-color flow cytometric analysis of Vγ2Vδ2 T cells in PBMC before (left panel) and after (right panel) 10 day stimulation is shown. ( B ) Inhibition of EJ-1 bladder carcinoma cell growth by exposure to compound 7 and γδ T cells. Compound 7 was added to cultures of EJ-1 tumor cells followed by the addition of Vγ2Vδ2 T cell to some cultures16 h later. Cell growth was assessed in a real-time cell analyzer system. Culture conditions were: (1) 50 nM compound 7 + medium, (2) 0 nM compound 7 + γδ T cells, (3) 1.56 nM compound 7 + γδ T cells, (4) 12.5 nM compound 7 + γδ T cells, (5) 25 nM compound 7 + γδ T cells, (6) 50 nM compound 7 + γδ T cells. ( C ) Degranulation of γδ T cells in response to U937 histocytoma pretreated with compound 7 . The proportion of CD107a + degranulated Vδ2 + cells were plotted against the concentrations of compound 7 used for the pretreatment of U937 cells. ( D ) Stimulation of IFN-γ production by Vγ2Vδ2 T cells by compound 7 . PBMC from healthy donor were cultured with 1 μM compound 7 . After 48 h, the culture supernatants were removed and IFN-γ levels determined by ELISA.
Figure Legend Snippet: Selective activation of Vγ2Vδ2 T cells by compound 7 . ( A ) Selective expansion of Vγ2Vδ2 T cells from blood αβ and γδ T cells after culture with compound 7 . PBMC from a prostate cancer patient were stimulated with 1 μM compound 7 and IL-2. The two-color flow cytometric analysis of Vγ2Vδ2 T cells in PBMC before (left panel) and after (right panel) 10 day stimulation is shown. ( B ) Inhibition of EJ-1 bladder carcinoma cell growth by exposure to compound 7 and γδ T cells. Compound 7 was added to cultures of EJ-1 tumor cells followed by the addition of Vγ2Vδ2 T cell to some cultures16 h later. Cell growth was assessed in a real-time cell analyzer system. Culture conditions were: (1) 50 nM compound 7 + medium, (2) 0 nM compound 7 + γδ T cells, (3) 1.56 nM compound 7 + γδ T cells, (4) 12.5 nM compound 7 + γδ T cells, (5) 25 nM compound 7 + γδ T cells, (6) 50 nM compound 7 + γδ T cells. ( C ) Degranulation of γδ T cells in response to U937 histocytoma pretreated with compound 7 . The proportion of CD107a + degranulated Vδ2 + cells were plotted against the concentrations of compound 7 used for the pretreatment of U937 cells. ( D ) Stimulation of IFN-γ production by Vγ2Vδ2 T cells by compound 7 . PBMC from healthy donor were cultured with 1 μM compound 7 . After 48 h, the culture supernatants were removed and IFN-γ levels determined by ELISA.

Techniques Used: Activation Assay, Flow Cytometry, Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay

Combination therapy with compound 7 and γδ T cells prolongs survival of immunodeficient NOG mice inoculated with either human EJ-1 bladder carcinoma cells or human HT1080 fibrosarcoma cells. On day 0, NOG mice were inoculated i.p. with 1.0 × 10 6 EJ-1 or 1.0 × 10 6 HT1080 tumor cells that had been stably transfected with the luc2 luciferase. On day 3 and day 6, the mice were treated with either (1) PBS (⚪), (2) compound 7 (∆) (2 μg (97 μg/kg) for EJ-1 or 10 μg (485 μg/kg) for HT1080), (3) 2 × 10 7 γδ T cells (▴), or (4) 2 × 10 7 γδ T cells and compound 7 (⚫) (2 μg (97 μg/kg) for EJ-1 or 10 μg (485 μg/kg) for HT1080). This treatment regimen was repeated for the duration of the experiment. ( A ) Survival of mice inoculated with EJ-1 cancer cells untreated or treated as above. Pooled data from two experiments performed identically is shown, n = 8 mice per group. ( B ) Survival of mice inoculated with HT1080 cancer cells untreated or treated as above. Pooled data from two experiments performed identically is shown, n = 8 mice per group except for mice treated with compound 7 where 4 mice were used. γδ T cells expressing Vγ2 Vδ2 TCRs (95–98% of transferred cells) were expanded from PBMC from a single donor and frozen for later use. Significance is shown for the survival difference between γδ T cells and compound 7 compared with γδ T cells alone as determined by the log-rank test.
Figure Legend Snippet: Combination therapy with compound 7 and γδ T cells prolongs survival of immunodeficient NOG mice inoculated with either human EJ-1 bladder carcinoma cells or human HT1080 fibrosarcoma cells. On day 0, NOG mice were inoculated i.p. with 1.0 × 10 6 EJ-1 or 1.0 × 10 6 HT1080 tumor cells that had been stably transfected with the luc2 luciferase. On day 3 and day 6, the mice were treated with either (1) PBS (⚪), (2) compound 7 (∆) (2 μg (97 μg/kg) for EJ-1 or 10 μg (485 μg/kg) for HT1080), (3) 2 × 10 7 γδ T cells (▴), or (4) 2 × 10 7 γδ T cells and compound 7 (⚫) (2 μg (97 μg/kg) for EJ-1 or 10 μg (485 μg/kg) for HT1080). This treatment regimen was repeated for the duration of the experiment. ( A ) Survival of mice inoculated with EJ-1 cancer cells untreated or treated as above. Pooled data from two experiments performed identically is shown, n = 8 mice per group. ( B ) Survival of mice inoculated with HT1080 cancer cells untreated or treated as above. Pooled data from two experiments performed identically is shown, n = 8 mice per group except for mice treated with compound 7 where 4 mice were used. γδ T cells expressing Vγ2 Vδ2 TCRs (95–98% of transferred cells) were expanded from PBMC from a single donor and frozen for later use. Significance is shown for the survival difference between γδ T cells and compound 7 compared with γδ T cells alone as determined by the log-rank test.

Techniques Used: Mouse Assay, Stable Transfection, Transfection, Luciferase, Expressing

4) Product Images from "Elevated Levels of Endocannabinoids in Chronic Hepatitis C May Modulate Cellular Immune Response and Hepatic Stellate Cell Activation"

Article Title: Elevated Levels of Endocannabinoids in Chronic Hepatitis C May Modulate Cellular Immune Response and Hepatic Stellate Cell Activation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms16047057

ECs affect inflammatory and fibrogenic hepatic cells activities. ( A ) Inflammatory cytokines mRNA and ( B ) CB1 and CB2 mRNA in human hepatocytes by 2.5 µM AEA and 20 µM 2-AG; ( C ) Fibrosis-related mRNA in hHSC and hHSC co-cultivated with PBMC from CHC patients by 2.5 µM AEA and 20 µM 2-AG. * p
Figure Legend Snippet: ECs affect inflammatory and fibrogenic hepatic cells activities. ( A ) Inflammatory cytokines mRNA and ( B ) CB1 and CB2 mRNA in human hepatocytes by 2.5 µM AEA and 20 µM 2-AG; ( C ) Fibrosis-related mRNA in hHSC and hHSC co-cultivated with PBMC from CHC patients by 2.5 µM AEA and 20 µM 2-AG. * p

Techniques Used:

Related Articles

Cell Culture:

Article Title: Treatment of primary HIV-1 infection with cyclosporin A coupled with highly active antiretroviral therapy
Article Snippet: PBMCs were thawed and resuspended at 37°C in RPMI 1640 medium (Life Technologies Inc., Gaithersburg, Maryland, USA) containing 2% inactivated AB human serum (Sigma Chemical Co.). .. PBMCs were plated at 2 × 105 cells/well in 96-well U-bottom cell culture plates (Corning-Costar Corp., Acton, Massachusetts, USA) and incubated with HIV-1 p24 gag protein (1 μg/well) or CMV lysates (1:10,000 final concentration) for 5 days. ..

Article Title: miR-125b-5p and miR-99a-5p downregulate human γδ T-cell activation and cytotoxicity
Article Snippet: γδ T-cell activation and expansion were performed as described previously., Briefly, 24-well plates were coated with 0.5 μg of anti-pan γδ TCR mAb (Immunotech, Beckman Coulter, Brea, CA, USA). .. After removal of this solution, PBMCs were added to the plates and cultured in RPMI 1640 medium (Corning, Corelle City, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL company, USA), 200 IU/ml recombinant human IL-2 (Beijing Read United Cross Pharmaceutical Co., Ltd., China), 100 mg/ml penicillin and 100 U/ml streptomycin at 37 °C and 5% CO2 for 5 days. ..

Incubation:

Article Title: Treatment of primary HIV-1 infection with cyclosporin A coupled with highly active antiretroviral therapy
Article Snippet: PBMCs were thawed and resuspended at 37°C in RPMI 1640 medium (Life Technologies Inc., Gaithersburg, Maryland, USA) containing 2% inactivated AB human serum (Sigma Chemical Co.). .. PBMCs were plated at 2 × 105 cells/well in 96-well U-bottom cell culture plates (Corning-Costar Corp., Acton, Massachusetts, USA) and incubated with HIV-1 p24 gag protein (1 μg/well) or CMV lysates (1:10,000 final concentration) for 5 days. ..

Concentration Assay:

Article Title: Treatment of primary HIV-1 infection with cyclosporin A coupled with highly active antiretroviral therapy
Article Snippet: PBMCs were thawed and resuspended at 37°C in RPMI 1640 medium (Life Technologies Inc., Gaithersburg, Maryland, USA) containing 2% inactivated AB human serum (Sigma Chemical Co.). .. PBMCs were plated at 2 × 105 cells/well in 96-well U-bottom cell culture plates (Corning-Costar Corp., Acton, Massachusetts, USA) and incubated with HIV-1 p24 gag protein (1 μg/well) or CMV lysates (1:10,000 final concentration) for 5 days. ..

Proliferation Assay:

Article Title: A dendritic cell receptor-targeted chimeric immunotherapeutic protein (C-HBV) for the treatment of chronic hepatitis B.
Article Snippet: The T cells were used either labeled with carboxyfluorescein N-succinimidyl ester (CFSE) (see below) or left unlabeled and added directly to cell cultures with antigen-loaded mature DCs. .. T cell proliferation assay Freshly isolated or previously frozen T cells from uninfected donors or PBMCs from chronic HBV carrier donors (in a 15 mL Tube, Corning, C352196) were washed once with D-PBS/5% autologous plasma (AP). .. The cells were resuspended at 4 × 106 cells/mL in D-PBS/5% autologous plasma (AP) and 110 μL of a 50 μM dilution of CFSE (Thermo Fisher Scientific, 65-0850-84) in D-PBS was added per 1 mL of cells.

Isolation:

Article Title: A dendritic cell receptor-targeted chimeric immunotherapeutic protein (C-HBV) for the treatment of chronic hepatitis B.
Article Snippet: The T cells were used either labeled with carboxyfluorescein N-succinimidyl ester (CFSE) (see below) or left unlabeled and added directly to cell cultures with antigen-loaded mature DCs. .. T cell proliferation assay Freshly isolated or previously frozen T cells from uninfected donors or PBMCs from chronic HBV carrier donors (in a 15 mL Tube, Corning, C352196) were washed once with D-PBS/5% autologous plasma (AP). .. The cells were resuspended at 4 × 106 cells/mL in D-PBS/5% autologous plasma (AP) and 110 μL of a 50 μM dilution of CFSE (Thermo Fisher Scientific, 65-0850-84) in D-PBS was added per 1 mL of cells.

Expressing:

Article Title: Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry
Article Snippet: .. PBMCs were thawed and washed with RPMI-1640 (Corning Cellgro) with 10% HI-FBS, 2mM L-Glutamine and antibiotics (penicillin [100 units/ml] and streptomycin [100μg/ml]) (Gibco BRL/Life technologies) and rested 37°C with 5% CO2 ) to sort cells based upon positive or negative expression of each marker. ..

Marker:

Article Title: Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry
Article Snippet: .. PBMCs were thawed and washed with RPMI-1640 (Corning Cellgro) with 10% HI-FBS, 2mM L-Glutamine and antibiotics (penicillin [100 units/ml] and streptomycin [100μg/ml]) (Gibco BRL/Life technologies) and rested 37°C with 5% CO2 ) to sort cells based upon positive or negative expression of each marker. ..

Recombinant:

Article Title: miR-125b-5p and miR-99a-5p downregulate human γδ T-cell activation and cytotoxicity
Article Snippet: γδ T-cell activation and expansion were performed as described previously., Briefly, 24-well plates were coated with 0.5 μg of anti-pan γδ TCR mAb (Immunotech, Beckman Coulter, Brea, CA, USA). .. After removal of this solution, PBMCs were added to the plates and cultured in RPMI 1640 medium (Corning, Corelle City, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL company, USA), 200 IU/ml recombinant human IL-2 (Beijing Read United Cross Pharmaceutical Co., Ltd., China), 100 mg/ml penicillin and 100 U/ml streptomycin at 37 °C and 5% CO2 for 5 days. ..

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    Corning Life Sciences blood mononuclear cells pbmcs
    LMP1 promoted NPC-induced MDSC differentiation and the expression of MDSC-related molecules in NPC cells. <t>CD33</t> + cells were isolated from healthy <t>PBMCs</t> using human CD33 MicroBeads and were co-cultured with NPC or NPC-LMP1 cells in a Transwell system for 48 h. The percentage of CD33 + CD11b + HLA-DR - MDSCs was measured by FACS staining. ( A ) Expression of the NLRP3, ASC, caspase-1, IL-1β, COX-2, Arg-1 and iNOS mRNAs was determined via real-time qRT-PCR, and the results were normalized against GAPDH expression. ( B ) NPC cells, including CNE2-vector, CNE2-LMP1, TW03-vector, and TW03-LMP1 cells, were cultured overnight following stimulation with LPS (0.1 μg/mL) and ATP (5 mM, 30 min) or no stimulation. Then, the culture supernatants were collected, and the concentrations of IL-1β, IL-6 and GM-CSF were measured by ELISA. ( C ) Representative density plots are shown as the CD33 + CD11b + cells in the HLA-DR - gate induced by NPC or NPC-LMP1 cells in different combinations. Representative data from 1 of 5 experiments are presented. ( D ) Statistical analysis of the percentage of MDSCs mediated by NPC cells in different combinations. CFSE-labeled PBMCs were co-cultured with M-MDSCs, CNE2-vector-MDSCs, CNE2-LMP1-MDSCs, TW03-vector-MDSCs or TW03-LMP1-MDSCs at a ratio of 1:1 in OKT3-coated 96-well plates. After 3 days, the cells were collected, stained with anti-human mAbs against CD4 and CD8 and quantified by flow cytometry. Representative FACS density plots ( E ) and the statistical data ( F ) showed that the proliferation of PBMCs and CD4 + and CD8 + T cells was markedly suppressed by CNE2-LMP1-MDSCs or TW03-LMP1-MDSCs compared with the effects of CNE2-vector-MDSCs or TW03-vector-MDSCs. Data are presented as the means ± SEM of representative experiments performed in triplicate. *P
    Blood Mononuclear Cells Pbmcs, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences peripheral blood mononuclear cell migration assay transwells
    Phosphorylation of ATM induces cytokine secretion and PBMC migration. a Quantification of CCL5, CXCL10, and IL16 secretion by ELISA following irradiation of BT549 breast cancer cells either in presence or absence of ATM inhibitor KU-55933 at 10 μM. Values plotted as mean ± std of duplicate spots. See Supplementary Fig. 7 . b Schematic for collection of conditioned media for PBMC <t>transwells.</t> Final concentration of ATM inhibitor KU-55933 was equal in all conditions (10 μM). c Migration of PBMCs towards tumor cell conditioned media from triple negative breast cancer cell lines MDA-MB-231 and BT-549 and luminal breast cancer cell lines ZR-75-1. Error bars represent ± standard deviation of duplicate runs. Significance determined by ANOVA using a Holm-Sidak post-hoc test
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    Corning Life Sciences pbmcs
    Nanoparticle formulation decreases curcumin cytotoxicity. <t>SUPT1</t> cells (Panel A) or stimulated <t>PBMCs</t> (Panel B) were exposed to increasing concentrations (1, 5, 10, 25, 50 and 100 µM) of sol-curcumin, nano-curcumin, azidothymidine (AZT) or nano-apotransferrin (10, 50 and 100 µg) for 16 h, after which cell viability was determined by MTT assay. PBMCs were cultured in the presence of IL-2 (20 IU/ml). Cell viability in the absence of drug was defined as 0% cytotoxicity. Error bars indicate SD. ** P ≤0.01, and ***, P ≤0.001 compared to nano-curcumin. * indicates µg apotransferrin protein that carry equivalent molar concentration of the drug.
    Pbmcs, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences flow cytometry peripheral blood mononuclear cells pbmcs
    Representative flow <t>cytometry</t> plots for one patient are shown and gating of CD3 + CD4 + ISOPE is indicated (a). Phorbol 12-myristate 13-acetate and ionomycin-stimulated peripheral blood mononuclear cells were stained and analyzed for IL-17 production (b). Inhibitory effect of Zn on IL-17 production with 3 μ mol/L concentration (c) and 30 μ mol/L concentration (d). Vitamin D3 effect on IL-17 production with 50 ng/mL concentration (e) and 500 ng/mL concentration (f).
    Flow Cytometry Peripheral Blood Mononuclear Cells Pbmcs, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LMP1 promoted NPC-induced MDSC differentiation and the expression of MDSC-related molecules in NPC cells. CD33 + cells were isolated from healthy PBMCs using human CD33 MicroBeads and were co-cultured with NPC or NPC-LMP1 cells in a Transwell system for 48 h. The percentage of CD33 + CD11b + HLA-DR - MDSCs was measured by FACS staining. ( A ) Expression of the NLRP3, ASC, caspase-1, IL-1β, COX-2, Arg-1 and iNOS mRNAs was determined via real-time qRT-PCR, and the results were normalized against GAPDH expression. ( B ) NPC cells, including CNE2-vector, CNE2-LMP1, TW03-vector, and TW03-LMP1 cells, were cultured overnight following stimulation with LPS (0.1 μg/mL) and ATP (5 mM, 30 min) or no stimulation. Then, the culture supernatants were collected, and the concentrations of IL-1β, IL-6 and GM-CSF were measured by ELISA. ( C ) Representative density plots are shown as the CD33 + CD11b + cells in the HLA-DR - gate induced by NPC or NPC-LMP1 cells in different combinations. Representative data from 1 of 5 experiments are presented. ( D ) Statistical analysis of the percentage of MDSCs mediated by NPC cells in different combinations. CFSE-labeled PBMCs were co-cultured with M-MDSCs, CNE2-vector-MDSCs, CNE2-LMP1-MDSCs, TW03-vector-MDSCs or TW03-LMP1-MDSCs at a ratio of 1:1 in OKT3-coated 96-well plates. After 3 days, the cells were collected, stained with anti-human mAbs against CD4 and CD8 and quantified by flow cytometry. Representative FACS density plots ( E ) and the statistical data ( F ) showed that the proliferation of PBMCs and CD4 + and CD8 + T cells was markedly suppressed by CNE2-LMP1-MDSCs or TW03-LMP1-MDSCs compared with the effects of CNE2-vector-MDSCs or TW03-vector-MDSCs. Data are presented as the means ± SEM of representative experiments performed in triplicate. *P

    Journal: PLoS Pathogens

    Article Title: LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma

    doi: 10.1371/journal.ppat.1006503

    Figure Lengend Snippet: LMP1 promoted NPC-induced MDSC differentiation and the expression of MDSC-related molecules in NPC cells. CD33 + cells were isolated from healthy PBMCs using human CD33 MicroBeads and were co-cultured with NPC or NPC-LMP1 cells in a Transwell system for 48 h. The percentage of CD33 + CD11b + HLA-DR - MDSCs was measured by FACS staining. ( A ) Expression of the NLRP3, ASC, caspase-1, IL-1β, COX-2, Arg-1 and iNOS mRNAs was determined via real-time qRT-PCR, and the results were normalized against GAPDH expression. ( B ) NPC cells, including CNE2-vector, CNE2-LMP1, TW03-vector, and TW03-LMP1 cells, were cultured overnight following stimulation with LPS (0.1 μg/mL) and ATP (5 mM, 30 min) or no stimulation. Then, the culture supernatants were collected, and the concentrations of IL-1β, IL-6 and GM-CSF were measured by ELISA. ( C ) Representative density plots are shown as the CD33 + CD11b + cells in the HLA-DR - gate induced by NPC or NPC-LMP1 cells in different combinations. Representative data from 1 of 5 experiments are presented. ( D ) Statistical analysis of the percentage of MDSCs mediated by NPC cells in different combinations. CFSE-labeled PBMCs were co-cultured with M-MDSCs, CNE2-vector-MDSCs, CNE2-LMP1-MDSCs, TW03-vector-MDSCs or TW03-LMP1-MDSCs at a ratio of 1:1 in OKT3-coated 96-well plates. After 3 days, the cells were collected, stained with anti-human mAbs against CD4 and CD8 and quantified by flow cytometry. Representative FACS density plots ( E ) and the statistical data ( F ) showed that the proliferation of PBMCs and CD4 + and CD8 + T cells was markedly suppressed by CNE2-LMP1-MDSCs or TW03-LMP1-MDSCs compared with the effects of CNE2-vector-MDSCs or TW03-vector-MDSCs. Data are presented as the means ± SEM of representative experiments performed in triplicate. *P

    Article Snippet: Tumor-associated MDSC induction in vitro Tumor-associated MDSCs were generated from CD33+ cells isolated by anti-CD33 beads (Miltenyi Biotec Company) from peripheral blood mononuclear cells (PBMCs) of healthy donors in a co-culture Transwell system (0.4-μm pore, Corning) with the NPC cell lines TW03, CNE2, TW03-LMP1 or CNE2-LMP1 as previously described.

    Techniques: Expressing, Isolation, Cell Culture, FACS, Staining, Quantitative RT-PCR, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry, Cytometry

    Phosphorylation of ATM induces cytokine secretion and PBMC migration. a Quantification of CCL5, CXCL10, and IL16 secretion by ELISA following irradiation of BT549 breast cancer cells either in presence or absence of ATM inhibitor KU-55933 at 10 μM. Values plotted as mean ± std of duplicate spots. See Supplementary Fig. 7 . b Schematic for collection of conditioned media for PBMC transwells. Final concentration of ATM inhibitor KU-55933 was equal in all conditions (10 μM). c Migration of PBMCs towards tumor cell conditioned media from triple negative breast cancer cell lines MDA-MB-231 and BT-549 and luminal breast cancer cell lines ZR-75-1. Error bars represent ± standard deviation of duplicate runs. Significance determined by ANOVA using a Holm-Sidak post-hoc test

    Journal: Nature Communications

    Article Title: Multi-omics analysis reveals neoantigen-independent immune cell infiltration in copy-number driven cancers

    doi: 10.1038/s41467-018-03730-x

    Figure Lengend Snippet: Phosphorylation of ATM induces cytokine secretion and PBMC migration. a Quantification of CCL5, CXCL10, and IL16 secretion by ELISA following irradiation of BT549 breast cancer cells either in presence or absence of ATM inhibitor KU-55933 at 10 μM. Values plotted as mean ± std of duplicate spots. See Supplementary Fig. 7 . b Schematic for collection of conditioned media for PBMC transwells. Final concentration of ATM inhibitor KU-55933 was equal in all conditions (10 μM). c Migration of PBMCs towards tumor cell conditioned media from triple negative breast cancer cell lines MDA-MB-231 and BT-549 and luminal breast cancer cell lines ZR-75-1. Error bars represent ± standard deviation of duplicate runs. Significance determined by ANOVA using a Holm-Sidak post-hoc test

    Article Snippet: Peripheral blood mononuclear cell migration assay Transwells (6.5 mm, 3.0 μm pore size) were acquired from Corning (Kennebunk, ME) and equilibrated overnight in RPMI containing 2% FBS.

    Techniques: Migration, Enzyme-linked Immunosorbent Assay, Irradiation, Concentration Assay, Multiple Displacement Amplification, Standard Deviation

    Nanoparticle formulation decreases curcumin cytotoxicity. SUPT1 cells (Panel A) or stimulated PBMCs (Panel B) were exposed to increasing concentrations (1, 5, 10, 25, 50 and 100 µM) of sol-curcumin, nano-curcumin, azidothymidine (AZT) or nano-apotransferrin (10, 50 and 100 µg) for 16 h, after which cell viability was determined by MTT assay. PBMCs were cultured in the presence of IL-2 (20 IU/ml). Cell viability in the absence of drug was defined as 0% cytotoxicity. Error bars indicate SD. ** P ≤0.01, and ***, P ≤0.001 compared to nano-curcumin. * indicates µg apotransferrin protein that carry equivalent molar concentration of the drug.

    Journal: PLoS ONE

    Article Title: Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro

    doi: 10.1371/journal.pone.0023388

    Figure Lengend Snippet: Nanoparticle formulation decreases curcumin cytotoxicity. SUPT1 cells (Panel A) or stimulated PBMCs (Panel B) were exposed to increasing concentrations (1, 5, 10, 25, 50 and 100 µM) of sol-curcumin, nano-curcumin, azidothymidine (AZT) or nano-apotransferrin (10, 50 and 100 µg) for 16 h, after which cell viability was determined by MTT assay. PBMCs were cultured in the presence of IL-2 (20 IU/ml). Cell viability in the absence of drug was defined as 0% cytotoxicity. Error bars indicate SD. ** P ≤0.01, and ***, P ≤0.001 compared to nano-curcumin. * indicates µg apotransferrin protein that carry equivalent molar concentration of the drug.

    Article Snippet: Nanoparticle localization assay SUPT1 cells obtained from the NIH-AIDS Reference and Reagents Program were used . (1×106 ) SUPT1 cells or stimulated PBMCs were seeded in 30 mm dishes (Corning Lifesciences) and treated with curcumin, either soluble or incorporated into nanoparticles at 1 and 10 µM concentrations and the cells were incubated at different time points (1, 2, 4 and 6 h).

    Techniques: MTT Assay, Cell Culture, Concentration Assay

    Nano-curcumin more effectively inhibits HIV-1 replication through a mechanism dependent on transferrin receptor. A C) SUPT1 cells (Panel A) or stimulated PBMCs (Panel C) were challenged for 2 h with HIV-1 93IN101 (1 mg p24/ml) in the presence of increasing concentrations (1, 2.5, 5, 10, 20 and 30 µM) of sol-curcumin, nano-curcumin, or nano-apotransferrin (10 and 50 µg). They were then incubated for a further 96 h, after which viral replication was measured by p24 antigen capture assay. *indicates µg apotransferrin protein that carry equivalent molar concentration of the drug. B D) SUP-T1 cells or stimulated PBMCs were challenged for 2 h with HIV-1 93IN101 in the presence of 2.5 or 5.0 µM concentrations of sol-curcumin, nano-curcumin or nano-curcumin in the presence of transferrin receptor antibody (100 ng/ml). After 96 h incubation, viral replication was measured by p24 antigen capture assay. In both these experiments, viral replication in the absence of drug was defined as 0% inhibition; Azidothymidine (AZT) was employed as a positive control. Error bars indicate SD. ** P ≤0.01, and ***, P ≤0.001 compared to sol-curcumin; n.s.: non-significant.

    Journal: PLoS ONE

    Article Title: Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro

    doi: 10.1371/journal.pone.0023388

    Figure Lengend Snippet: Nano-curcumin more effectively inhibits HIV-1 replication through a mechanism dependent on transferrin receptor. A C) SUPT1 cells (Panel A) or stimulated PBMCs (Panel C) were challenged for 2 h with HIV-1 93IN101 (1 mg p24/ml) in the presence of increasing concentrations (1, 2.5, 5, 10, 20 and 30 µM) of sol-curcumin, nano-curcumin, or nano-apotransferrin (10 and 50 µg). They were then incubated for a further 96 h, after which viral replication was measured by p24 antigen capture assay. *indicates µg apotransferrin protein that carry equivalent molar concentration of the drug. B D) SUP-T1 cells or stimulated PBMCs were challenged for 2 h with HIV-1 93IN101 in the presence of 2.5 or 5.0 µM concentrations of sol-curcumin, nano-curcumin or nano-curcumin in the presence of transferrin receptor antibody (100 ng/ml). After 96 h incubation, viral replication was measured by p24 antigen capture assay. In both these experiments, viral replication in the absence of drug was defined as 0% inhibition; Azidothymidine (AZT) was employed as a positive control. Error bars indicate SD. ** P ≤0.01, and ***, P ≤0.001 compared to sol-curcumin; n.s.: non-significant.

    Article Snippet: Nanoparticle localization assay SUPT1 cells obtained from the NIH-AIDS Reference and Reagents Program were used . (1×106 ) SUPT1 cells or stimulated PBMCs were seeded in 30 mm dishes (Corning Lifesciences) and treated with curcumin, either soluble or incorporated into nanoparticles at 1 and 10 µM concentrations and the cells were incubated at different time points (1, 2, 4 and 6 h).

    Techniques: Incubation, Concentration Assay, Inhibition, Positive Control

    Nanoparticle formulation increases curcumin uptake, which is inhibited by transferrin receptor blockade. A) SUP-T1 cells were incubated for 1 h with curcumin formulations as indicated, then examined by confocal microscopy. (i) Cells without curcumin; (ii) 1 µM sol-curcumin; (iii) 1 µM nano-curcumin; or (iv) 1 µM nano-curcumin in the presence of transferrin receptor antibody (100 ng/ml). Each panel contains three images: fluorescence, bright field and merged. B) SUP-T1 cells (i) or stimulated PBMCs (ii) were incubated for 1 h with curcumin formulations, after which intrinsic fluorescence of intracellular curcumin was determined quantitatively by fluorometric analysis. Cells were treated with 5 µM sol-curcumin, 5 µM nano-curcumin, or 5 µM nano-curcumin in the presence of antibodies to the transferrin receptor (TrR-Ab; 100 ng/ml). All the values are normalized to that obtained from SUPT1 cells. Error bars indicate standard deviation (SD). ***, P ≤0.001 compared to sol-curcumin; n.s.: non-significant.

    Journal: PLoS ONE

    Article Title: Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro

    doi: 10.1371/journal.pone.0023388

    Figure Lengend Snippet: Nanoparticle formulation increases curcumin uptake, which is inhibited by transferrin receptor blockade. A) SUP-T1 cells were incubated for 1 h with curcumin formulations as indicated, then examined by confocal microscopy. (i) Cells without curcumin; (ii) 1 µM sol-curcumin; (iii) 1 µM nano-curcumin; or (iv) 1 µM nano-curcumin in the presence of transferrin receptor antibody (100 ng/ml). Each panel contains three images: fluorescence, bright field and merged. B) SUP-T1 cells (i) or stimulated PBMCs (ii) were incubated for 1 h with curcumin formulations, after which intrinsic fluorescence of intracellular curcumin was determined quantitatively by fluorometric analysis. Cells were treated with 5 µM sol-curcumin, 5 µM nano-curcumin, or 5 µM nano-curcumin in the presence of antibodies to the transferrin receptor (TrR-Ab; 100 ng/ml). All the values are normalized to that obtained from SUPT1 cells. Error bars indicate standard deviation (SD). ***, P ≤0.001 compared to sol-curcumin; n.s.: non-significant.

    Article Snippet: Nanoparticle localization assay SUPT1 cells obtained from the NIH-AIDS Reference and Reagents Program were used . (1×106 ) SUPT1 cells or stimulated PBMCs were seeded in 30 mm dishes (Corning Lifesciences) and treated with curcumin, either soluble or incorporated into nanoparticles at 1 and 10 µM concentrations and the cells were incubated at different time points (1, 2, 4 and 6 h).

    Techniques: Incubation, Confocal Microscopy, Fluorescence, Standard Deviation

    Representative flow cytometry plots for one patient are shown and gating of CD3 + CD4 + ISOPE is indicated (a). Phorbol 12-myristate 13-acetate and ionomycin-stimulated peripheral blood mononuclear cells were stained and analyzed for IL-17 production (b). Inhibitory effect of Zn on IL-17 production with 3 μ mol/L concentration (c) and 30 μ mol/L concentration (d). Vitamin D3 effect on IL-17 production with 50 ng/mL concentration (e) and 500 ng/mL concentration (f).

    Journal: Journal of Immunology Research

    Article Title: The Interplay between Zinc, Vitamin D and, IL-17 in Patients with Chronic Hepatitis C Liver Disease

    doi: 10.1155/2015/846348

    Figure Lengend Snippet: Representative flow cytometry plots for one patient are shown and gating of CD3 + CD4 + ISOPE is indicated (a). Phorbol 12-myristate 13-acetate and ionomycin-stimulated peripheral blood mononuclear cells were stained and analyzed for IL-17 production (b). Inhibitory effect of Zn on IL-17 production with 3 μ mol/L concentration (c) and 30 μ mol/L concentration (d). Vitamin D3 effect on IL-17 production with 50 ng/mL concentration (e) and 500 ng/mL concentration (f).

    Article Snippet: Intracellular Cytokine Staining and Flow Cytometry Peripheral blood mononuclear cells (PBMCs) from patients were cultured at a concentration of 5 × 105 /well in 200 μ L of complete medium in 96-well U bottom cell culture plates (Corning Incorporated, Corning, NY) and stimulated with 10 ng/mL of PMA plus 1 μ g/mL IO, in the presence or absence of vitamin D3 in two different concentrations (low 50 ng/mL and high 500 ng/mL) in some wells or Zn in the form of zinc sulphate in both low and high conc. (3 μ mol/L and 30 μ mol/L.) in other wells.

    Techniques: Flow Cytometry, Cytometry, Staining, Concentration Assay