pbmcs  (Millipore)

Journal: Stem Cells Translational Medicine

Article Title: A Proinflammatory Secretome Mediates the Impaired Immunopotency of Human Mesenchymal Stromal Cells in Elderly Patients with Atherosclerosis

doi: 10.1002/sctm.16-0221

Figure Lengend Snippet: MSCs from pericardial and subcutaneous adipose tissue equally suppress T‐cell proliferation. (A) : Representative example of a flow cytometry proliferation analysis of monocyte depleted peripheral blood mononuclear cells in coculture with subcutaneous or pericardial MSCs. MSCs from subcutaneous and pericardial fat have similar ability to suppress activated T‐cells' proliferation (B) and to support T‐cell viability (C) ( n = 8). Abbreviations: 7 AAD, 7‐aminoactinomycin D; adMSCs, adipose tissue‐derived MSCs; EI, expansion index; CFSE, carboxyfluorescein succinimidyl ester; FSC‐A: forward scatter area; SSC‐A: side scatter area; SSC‐H: side scatter height; SSC‐W: side scatter width.

Article Snippet: To assess the effect of MSCs on suppressing monocyte‐depleted PBMCs proliferation, PBMCs were labeled with 10 uM carboxyfluorescein succinimidyl ester (CFSE) (Sigma), stimulated with anti‐CD3/CD28 beads (1 bead per cell) (Dynabeads Human T‐Activator CD3/CD28, Life Technologies) and cultured for 4 days with MSCs.

Techniques: Flow Cytometry, Cytometry, Derivative Assay

Journal: PLoS ONE

Article Title: Raman Spectroscopy characterization extracellular vesicles from bovine placenta and peripheral blood mononuclear cells

doi: 10.1371/journal.pone.0235214

Figure Lengend Snippet: Raman fingerprints of extracellular vesicles. Raman spectra of trophoblast (a) and peripheral mononuclear blood cell (PBMC) (b) extracellular vesicles, n = 30–35 . The spectral shift positions are highlighted in cyan color and indicated by red numbers.

Article Snippet: Peripheral blood mononuclear cells were isolated by density gradient centrifugation (Accu-Paque, Accurate Chem.

Techniques:

Journal: PLoS ONE

Article Title: Raman Spectroscopy characterization extracellular vesicles from bovine placenta and peripheral blood mononuclear cells

doi: 10.1371/journal.pone.0235214

Figure Lengend Snippet: PCA and LDA analyses of the extracellular vesicles derived from peripheral mononuclear blood cells (P001, P002 and P003) and trophoblast cells (T001, T002 and T003). Scatter plot of the PCA results show the PC1 and PC2 scores assigned to each spectrum ( N = 90 ) (a). Linear discriminant analysis, the first 10 PC loadings calculated by means of PCA were used for LDA. Each spot represents a single spectrum. The crosses indicate the mean canonical observation score obtained for each group ( N = 90 ) (b). 2D PCA (c) and LDA (d) plots of PBMC-derived vesicles from three different cows, and 2D PCA (e) and LDA (f) plots of trophoblast-derived vesicles from three different animal individuals.

Article Snippet: Peripheral blood mononuclear cells were isolated by density gradient centrifugation (Accu-Paque, Accurate Chem.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Raman Spectroscopy characterization extracellular vesicles from bovine placenta and peripheral blood mononuclear cells

doi: 10.1371/journal.pone.0235214

Figure Lengend Snippet: Principal component analysis (PCA) loading plots corresponding to PC1, PC2, and PC3 from the data of Fig 6A for extracellular vesicles derived from peripheral mononuclear blood cells and trophoblast cells.

Article Snippet: Peripheral blood mononuclear cells were isolated by density gradient centrifugation (Accu-Paque, Accurate Chem.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Raman Spectroscopy characterization extracellular vesicles from bovine placenta and peripheral blood mononuclear cells

doi: 10.1371/journal.pone.0235214

Figure Lengend Snippet: Raman fingerprint of extracellular vesicles (EVs) isolated from peripheral mononuclear blood cells (PBMC) and trophoblast cells. The solid line indicates the average of 90 spectra ± 1 standard deviation (shaded grey areas). The areas highlighted in cyan color showed the intensity differences. The peaks marked in red only exist in PBMC-derived EVs, while the peaks in blue only exist in trophoblast-derived EVs (a). Mean Raman peak intensity analysis of PBMC and trophoblast-derived EVs, N = 90 , * means P

Article Snippet: Peripheral blood mononuclear cells were isolated by density gradient centrifugation (Accu-Paque, Accurate Chem.

Techniques: Isolation, Standard Deviation, Derivative Assay

Journal: Cell Proliferation

Article Title: Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor, et al. Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor

doi: 10.1111/cpr.12858

Figure Lengend Snippet: Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry

Article Snippet: Peripheral blood mononuclear cells were isolated from the whole blood of healthy donors by Ficoll density gradient centrifugation.

Techniques: Isolation, Labeling, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining, Flow Cytometry

Journal: Antimicrobial Agents and Chemotherapy

Article Title: MIV-150/Zinc Acetate Gel Inhibits Cell-Associated Simian-Human Immunodeficiency Virus Reverse Transcriptase Infection in a Macaque Vaginal Explant Model

doi: 10.1128/AAC.00073-15

Figure Lengend Snippet: Infection of macaque vaginal tissue after challenge with cell-associated SHIV-RT. Explants were prepared from vaginal biopsy specimens of SHIV-exposed uninfected animals ( n = 5). PHA/IL-2-stimulated explants were incubated with mitomycin-C-treated SHIV-RT-infected PBMCs (50 to 5 × 10 4 cells/explant; 3 to 4 explants/donor/condition) in 96-well plates for 18 h. Then the tissues were washed and cultured for 11 days in the presence of IL-2. PBMCs (5 × 10 4 ) cultured alone (PBMCs only) in the presence of IL-2 served as controls. SIV p27 release was measured at 0, 3, 7, and 11 days of culture by p27 ELISA. (A) Shown is the summary from 3 to 5 independent experiments using 10 2 to 10 4 infected PBMCs (means ± SEM) and an individual experiment using 50 and 5 × 10 4 cells (means ± SEM of replicate wells under each condition). (B) Shown are log 10 -transformed SOFT and CUM analyses (days 3 to 11 of culture) of the experiments presented in panel A. The LLOQ of the assay is denoted for both SOFT (solid line) and CUM (dotted line).

Article Snippet: Tissues were washed twice in PBS, cut using an Acu-Punch (Acuderm, Fort Lauderdale, FL) to ∼3- by 3-mm pieces, and cultured in 96-well plates in the presence of 5 μg/ml PHA and 100 U/ml IL-2 in cDMEM (Dulbecco's modified Eagle's medium [DMEM] [Cellgro Mediatech] supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM minimal essential medium [MEM] nonessential amino acids [Irvine Scientific, Santa Ana, CA]) for 48 h. Thawed SHIV-RT-infected PBMCs were cultured overnight with 50 U/ml IL-2 in R10, incubated with 200 μg/ml mitomycin C (Sigma-Aldrich) for 1 h, and washed (3 times) in PBS before the coculture was set up.

Techniques: Infection, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: MIV-150/Zinc Acetate Gel Inhibits Cell-Associated Simian-Human Immunodeficiency Virus Reverse Transcriptase Infection in a Macaque Vaginal Explant Model

doi: 10.1128/AAC.00073-15

Figure Lengend Snippet: De novo viral production in SHIV-RT-infected PBMCs is inhibited by mitomycin-C. Infected PBMCs were thawed, cultured overnight in the presence of IL-2, and then treated with mitomycin-C (versus untreated control PBMCs) and cultured for 14 days in the presence of IL-2 ( n = 3 replicates per condition). Supernatants were collected at days 0 (18 h after culture setup), 3, 7, 11, and 14, and replicates were pooled for analysis by qRT-PCR. Shown are the SIV gag copies per milliliter from two independent experiments.

Article Snippet: Tissues were washed twice in PBS, cut using an Acu-Punch (Acuderm, Fort Lauderdale, FL) to ∼3- by 3-mm pieces, and cultured in 96-well plates in the presence of 5 μg/ml PHA and 100 U/ml IL-2 in cDMEM (Dulbecco's modified Eagle's medium [DMEM] [Cellgro Mediatech] supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM minimal essential medium [MEM] nonessential amino acids [Irvine Scientific, Santa Ana, CA]) for 48 h. Thawed SHIV-RT-infected PBMCs were cultured overnight with 50 U/ml IL-2 in R10, incubated with 200 μg/ml mitomycin C (Sigma-Aldrich) for 1 h, and washed (3 times) in PBS before the coculture was set up.

Techniques: Infection, Cell Culture, Quantitative RT-PCR

Journal: Antimicrobial Agents and Chemotherapy

Article Title: MIV-150/Zinc Acetate Gel Inhibits Cell-Associated Simian-Human Immunodeficiency Virus Reverse Transcriptase Infection in a Macaque Vaginal Explant Model

doi: 10.1128/AAC.00073-15

Figure Lengend Snippet: Comparison of vaginal tissue infection after challenge with cell-associated SHIV-RT in contact-dependent and -independent models. Explants were prepared from vaginal biopsy or necropsy tissues of naive ( n = 2) and SHIV-exposed uninfected ( n = 2) animals. PHA/IL-2-stimulated explants were placed in the bottom chamber of a 12-well Transwell plate with 0.4-μm-pore inserts ( n = 4 explants per well/donor/condition). SHIV-RT-infected, mitomycin C-treated PBMCs (10 3 cells per explant) were added to the bottom chamber of the Transwell plate with tissues (Contact) or on the top of the insert (No Contact). Cultures were maintained for 14 days in the presence of IL-2. SIV p27 release in the bottom chamber was measured at 0 (18 h after culture setup), 3, 7, 11, and 14 days of culture by p27 ELISA. Shown are individual experiments (A) and the summaries from the SOFT and CUM analyses (B) (means ± SEM) of the experiments presented in panel A. The LLOQs of the assay are denoted as dotted lines in panel A. The LLOQs for both SOFT (solid line) and CUM (dotted line) are indicated in panel B.

Article Snippet: Tissues were washed twice in PBS, cut using an Acu-Punch (Acuderm, Fort Lauderdale, FL) to ∼3- by 3-mm pieces, and cultured in 96-well plates in the presence of 5 μg/ml PHA and 100 U/ml IL-2 in cDMEM (Dulbecco's modified Eagle's medium [DMEM] [Cellgro Mediatech] supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM minimal essential medium [MEM] nonessential amino acids [Irvine Scientific, Santa Ana, CA]) for 48 h. Thawed SHIV-RT-infected PBMCs were cultured overnight with 50 U/ml IL-2 in R10, incubated with 200 μg/ml mitomycin C (Sigma-Aldrich) for 1 h, and washed (3 times) in PBS before the coculture was set up.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: MIV-150/Zinc Acetate Gel Inhibits Cell-Associated Simian-Human Immunodeficiency Virus Reverse Transcriptase Infection in a Macaque Vaginal Explant Model

doi: 10.1128/AAC.00073-15

Figure Lengend Snippet: Phenotype of SHIV-RT-infected PBMCs. Uninfected control (A) and SHIV-RT-infected (B to D) PBMCs were thawed and cultured overnight in the presence of IL-2. Then PBMCs were incubated with Live/Dead Aqua stain and anti-CD3 (Alexa Fluor 700), anti-CD4 (PerCP-Cy5.5), anti-CD95 (V450), and anti-CD28 APC antibodies followed by intracellular staining with anti-p27 RPE antibody. Mononuclear cells were gated on the total live (Aqua − ) population. The gating strategy is presented in B to D. First, CD3 − p27 + and CD3 + p27 + T cells were gated (B). Next, CD4 + T cells (C) were gated within CD3 + p27 + T cells. Then, infected T cell subsets were identified within CD3 + CD4 + p27 + T cells (D).

Article Snippet: Tissues were washed twice in PBS, cut using an Acu-Punch (Acuderm, Fort Lauderdale, FL) to ∼3- by 3-mm pieces, and cultured in 96-well plates in the presence of 5 μg/ml PHA and 100 U/ml IL-2 in cDMEM (Dulbecco's modified Eagle's medium [DMEM] [Cellgro Mediatech] supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM minimal essential medium [MEM] nonessential amino acids [Irvine Scientific, Santa Ana, CA]) for 48 h. Thawed SHIV-RT-infected PBMCs were cultured overnight with 50 U/ml IL-2 in R10, incubated with 200 μg/ml mitomycin C (Sigma-Aldrich) for 1 h, and washed (3 times) in PBS before the coculture was set up.

Techniques: Infection, Cell Culture, Incubation, Staining

Journal: Antimicrobial Agents and Chemotherapy

Article Title: MIV-150/Zinc Acetate Gel Inhibits Cell-Associated Simian-Human Immunodeficiency Virus Reverse Transcriptase Infection in a Macaque Vaginal Explant Model

doi: 10.1128/AAC.00073-15

Figure Lengend Snippet: Characterization of cell-associated SHIV-RT stock. (A) Total DNA and RNA were isolated from 10 6 infected PBMCs. SIV gag qPCR and qRT-PCR were run using 50 μg of DNA and RNA as the templates, respectively. Shown are calculated per cell SIV gag copies (single experiment). (B) Infected PBMCs were thawed and immediately used in the p27 ELISA. Shown is p27 content in lysed PBMCs (single experiment).

Article Snippet: Tissues were washed twice in PBS, cut using an Acu-Punch (Acuderm, Fort Lauderdale, FL) to ∼3- by 3-mm pieces, and cultured in 96-well plates in the presence of 5 μg/ml PHA and 100 U/ml IL-2 in cDMEM (Dulbecco's modified Eagle's medium [DMEM] [Cellgro Mediatech] supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM minimal essential medium [MEM] nonessential amino acids [Irvine Scientific, Santa Ana, CA]) for 48 h. Thawed SHIV-RT-infected PBMCs were cultured overnight with 50 U/ml IL-2 in R10, incubated with 200 μg/ml mitomycin C (Sigma-Aldrich) for 1 h, and washed (3 times) in PBS before the coculture was set up.

Techniques: Isolation, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: MIV-150/Zinc Acetate Gel Inhibits Cell-Associated Simian-Human Immunodeficiency Virus Reverse Transcriptase Infection in a Macaque Vaginal Explant Model

doi: 10.1128/AAC.00073-15

Figure Lengend Snippet: MZC inhibits ex vivo cell-associated SHIV-RT infection up to 4 days after gel exposure. Explants were prepared from vaginal biopsy or necropsy tissues of SHIV-RT-exposed uninfected ( n = 5, 24-h group), naive ( n = 3, 4-day group), and SHIV-RT-infected animals ( n = 2, 24-h group and n = 2, 4-day group). PHA/IL-2-stimulated explants were challenged with cell-associated SHIV-RT (10 3 infected PBMCs per explant; 3 to 4 explants/donor/condition) in 96-well plates 24 h or 4 days after exposure to diluted gels. Shown are log 10 -transformed SOFT and CUM analyses (means ± SEM; days 3 to 14 of culture). Summaries from 5 to 7 independent experiments (24 h) and 5 independent experiments (4 days) are shown. The LLOQs of the assay are denoted for both SOFT (solid line) and CUM (dotted line). *, P

Article Snippet: Tissues were washed twice in PBS, cut using an Acu-Punch (Acuderm, Fort Lauderdale, FL) to ∼3- by 3-mm pieces, and cultured in 96-well plates in the presence of 5 μg/ml PHA and 100 U/ml IL-2 in cDMEM (Dulbecco's modified Eagle's medium [DMEM] [Cellgro Mediatech] supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM minimal essential medium [MEM] nonessential amino acids [Irvine Scientific, Santa Ana, CA]) for 48 h. Thawed SHIV-RT-infected PBMCs were cultured overnight with 50 U/ml IL-2 in R10, incubated with 200 μg/ml mitomycin C (Sigma-Aldrich) for 1 h, and washed (3 times) in PBS before the coculture was set up.

Techniques: Ex Vivo, Infection, Transformation Assay