Structured Review

Becton Dickinson peripheral blood mononuclear cells pbmcs
Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts. (A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in <t>3′-RACE</t> experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells <t>(PBMCs),</t> kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A . (A) n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.
Peripheral Blood Mononuclear Cells Pbmcs, supplied by Becton Dickinson, 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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1) Product Images from "Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels"

Article Title: Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035461

Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts. (A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in 3′-RACE experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells (PBMCs), kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A . (A) n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.
Figure Legend Snippet: Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts. (A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in 3′-RACE experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells (PBMCs), kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A . (A) n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.

Techniques Used: Sequencing, Agarose Gel Electrophoresis, Nested PCR, Amplification, Molecular Weight, Marker, Clone Assay, Isolation

2) Product Images from "Zim CHIC: A cohort study of immune changes in the female genital tract associated with initiation and use of contraceptives, et al. Zim CHIC: A cohort study of immune changes in the female genital tract associated with initiation and use of contraceptives"

Article Title: Zim CHIC: A cohort study of immune changes in the female genital tract associated with initiation and use of contraceptives, et al. Zim CHIC: A cohort study of immune changes in the female genital tract associated with initiation and use of contraceptives

Journal: American Journal of Reproductive Immunology

doi: 10.1111/aji.13287

Flow cytometric gating strategy and baseline blood and cervical CD4 cells expressing CCR5 and CD69. Recovery of CD3 + CD4 + CCR5 + and CD3 + CD4 + CD69 + cells from (A) cervical cytobrush, (B) peripheral blood mononuclear cells (PBMC), (C) CD3 + CD4 + CCR5CD69 + double positives from cervical cytobrushes and PBMC (D) the proportion of CD3 + CD4 + CCR5 + and CD3 + CD4 + CD69 + from PBMC and cervical cytobrush demonstrating gating. Two senior laboratory scientists trained in advanced flow cytometry and masked to contraceptive group, independently reviewed and agreed upon gating parameters for each sample. The percent (D) and number (E) of CD3 + CD4 + CCR5 + and CD3 + CD4 + CD69 + cells in PBMC and cervical cytobrush samples collected from all evaluable participants at baseline, prior to initiation of any contraception. Mean values with SD are indicated with red bars and brackets, respectively
Figure Legend Snippet: Flow cytometric gating strategy and baseline blood and cervical CD4 cells expressing CCR5 and CD69. Recovery of CD3 + CD4 + CCR5 + and CD3 + CD4 + CD69 + cells from (A) cervical cytobrush, (B) peripheral blood mononuclear cells (PBMC), (C) CD3 + CD4 + CCR5CD69 + double positives from cervical cytobrushes and PBMC (D) the proportion of CD3 + CD4 + CCR5 + and CD3 + CD4 + CD69 + from PBMC and cervical cytobrush demonstrating gating. Two senior laboratory scientists trained in advanced flow cytometry and masked to contraceptive group, independently reviewed and agreed upon gating parameters for each sample. The percent (D) and number (E) of CD3 + CD4 + CCR5 + and CD3 + CD4 + CD69 + cells in PBMC and cervical cytobrush samples collected from all evaluable participants at baseline, prior to initiation of any contraception. Mean values with SD are indicated with red bars and brackets, respectively

Techniques Used: Expressing, Flow Cytometry

3) Product Images from "Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels"

Article Title: Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035461

Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts. (A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in 3′-RACE experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells (PBMCs), kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A . (A) n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.
Figure Legend Snippet: Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts. (A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in 3′-RACE experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells (PBMCs), kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A . (A) n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.

Techniques Used: Sequencing, Agarose Gel Electrophoresis, Nested PCR, Amplification, Molecular Weight, Marker, Clone Assay, Isolation

4) Product Images from "CD39+ Regulatory T Cells Attenuate Lipopolysaccharide-Induced Acute Lung Injury via Autophagy and the ERK/FOS Pathway"

Article Title: CD39+ Regulatory T Cells Attenuate Lipopolysaccharide-Induced Acute Lung Injury via Autophagy and the ERK/FOS Pathway

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.602605

The frequency of CD39 + Tregs are decreased in ARDS patients. (A) Representative FACS analysis shows CD39 + Tregs in the PBMCs in ARDS patients. (B) Analysis of CD4 + Foxp3 + CD39 + Tregs in PBMCs from ARDS patients and healthy donors. (C) Analysis of CD4 + Foxp3 + CD39 + Tregs in different groups of ARDS patients. ** P
Figure Legend Snippet: The frequency of CD39 + Tregs are decreased in ARDS patients. (A) Representative FACS analysis shows CD39 + Tregs in the PBMCs in ARDS patients. (B) Analysis of CD4 + Foxp3 + CD39 + Tregs in PBMCs from ARDS patients and healthy donors. (C) Analysis of CD4 + Foxp3 + CD39 + Tregs in different groups of ARDS patients. ** P

Techniques Used: FACS

5) Product Images from "Toll-Like Receptor 2-Mediated Innate Immune Responses against Junín Virus in Mice Lead to Antiviral Adaptive Immune Responses during Systemic Infection and Do Not Affect Viral Replication in the Brain"

Article Title: Toll-Like Receptor 2-Mediated Innate Immune Responses against Junín Virus in Mice Lead to Antiviral Adaptive Immune Responses during Systemic Infection and Do Not Affect Viral Replication in the Brain

Journal: Journal of Virology

doi: 10.1128/JVI.00050-14

Increased levels of Junín virus-specific CD8 + T cells but not humoral responses require TLR2 signaling. Mice were infected with JUNV C1 or LCMV. At 1 week postinfection, mouse PBMCs were collected and stained for NP-specific CD8 + T cells. (A and
Figure Legend Snippet: Increased levels of Junín virus-specific CD8 + T cells but not humoral responses require TLR2 signaling. Mice were infected with JUNV C1 or LCMV. At 1 week postinfection, mouse PBMCs were collected and stained for NP-specific CD8 + T cells. (A and

Techniques Used: Mouse Assay, Infection, Staining

Related Articles

Incubation:

Article Title: A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates
Article Snippet: Rhesus macaque PBMCs were incubated with 2 μg/ml DEN1-80E, DEN2-80E, DEN3-80E, DEN4-80E specific peptide pools (15 mer overlapping by 11, custom order JPT, Germany) or equal quantity of DMSO for non stimulated controls, in the presence of Brefeldin A and anti-human CD28 & CD49d antibodies (BD Biosciences, CA). .. 6 hrs post incubation at 37′c, PBMCs were surface stained for anti-mouse-CD3(BD #552852), CD4(BD #562402), CD8(BD #561421) followed by permealization & intracellular staining for anti-mouse IFNγ (BD #552882), IL-2 (BD #559334) and TNFα (BD #557647). .. Fixed samples were then run on FACS LSRII flow cytometer (BD Biosciences) and data was analyzed using FloJo software (Treestar Inc).

Article Title: Gene expression profile of peripheral blood mononuclear cells in response to HIV-VLPs stimulation
Article Snippet: In parallel, PBMCs isolated by Ficoll-Hypaque density gradient centrifugation from same normal healthy donors, were pulsed with same concentration of HIV-VLPs for 12 hours. .. Analysis of DC phenotype MDDCs and PBMCs were incubated for 30 min at 4°C with murine monoclonal antibodies specific for CD80, CD83, CD86, HLA-DR and CD14 [PBMCs] (BD Pharmingen, San Diego, CA), washed and then fixed with 2% paraformaldehyde for analysis with a FACScalibur flow cytometer (BD Pharmingen). ..

Article Title: Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome
Article Snippet: .. Heparinized peripheral blood was incubated in the dark at room temperature for 15 min with Peridinin chlorophyll protein-conjugated anti-CD45 (a PBMC marker) Ab (Becton Dickinson) and either phycoerythrin-conjugated anti-MCP Ab (anti-CD46, clone 122-2, Serotec), or phycoerythrin-conjugated isotype control Ab (Serotec). ..

Staining:

Article Title: A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates
Article Snippet: Rhesus macaque PBMCs were incubated with 2 μg/ml DEN1-80E, DEN2-80E, DEN3-80E, DEN4-80E specific peptide pools (15 mer overlapping by 11, custom order JPT, Germany) or equal quantity of DMSO for non stimulated controls, in the presence of Brefeldin A and anti-human CD28 & CD49d antibodies (BD Biosciences, CA). .. 6 hrs post incubation at 37′c, PBMCs were surface stained for anti-mouse-CD3(BD #552852), CD4(BD #562402), CD8(BD #561421) followed by permealization & intracellular staining for anti-mouse IFNγ (BD #552882), IL-2 (BD #559334) and TNFα (BD #557647). .. Fixed samples were then run on FACS LSRII flow cytometer (BD Biosciences) and data was analyzed using FloJo software (Treestar Inc).

Article Title: Multiple Sclerosis-Associated Changes in the Composition and Immune Functions of Spore-Forming Bacteria
Article Snippet: The FoxP3/transcription factor staining buffer set (eBioscience no. 00-5523-00) was used for staining of intracellular and intranuclear cytokines. .. The following antibodies were used for human PBMC staining: anti-CD3-PE.Cy7 (BD no. 563423), anti-CD4-PerCP.Cy5.5 (BioLegend no. 300530), anti-CD25-APC (BD no. 555434), anti-FoxP3-Alexa Fluor 488 (BD no. 560047) and anti-IL-10-PE (eBioscience no. 12-7108). .. Flow cytometry was performed on a BD Fortessa cell analyzer and results were analyzed using FlowJo software (TreeStar).

Article Title: Quantitative Characterization of the T Cell Receptor Repertoire of Naïve and Memory Subsets Using an Integrated Experimental and Computational Pipeline Which Is Robust, Economical, and Versatile
Article Snippet: CD4 positive selection using Miltenyi beads was followed by CD8 positive selection of the CD4 negative fraction following the manufacturers’ instructions. .. CD4+ and CD8+ PBMC were stained with the following antibodies (all BD biosciences): CD3-PE-Cy7, CD4-V450, CD8-AF700, CD45RA-PE-CF594, and CD27-APC, as well as the fixable nearIR live/dead cell stain (Invitrogen). .. Cells were gated on lymphocytes, singlets, live cells, CD3+ and either CD4+ or CD8+ and then sorted into naïve T cells (CD45RA high, CD27+), central memory (CM, CD45RA−, CD27+), effector memory (EM, CD45RA−, CD27−) and effector memory RA-expressing revertants (EMRA, CD45RA+, CD27−) on an Aria II (BD).

Article Title: Dynamic Changes of Th1 Cytokines and the Clinical Significance of the IFN-γ/TNF-α Ratio in Acute Brucellosis
Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus density-gradient media (Amersham Biosciences, Little Chalfont, UK) according to the manufacturer's protocol. .. Flow Cytometry and Cytometric Bead Array The PBMCs were harvested and stained with allophycocyanin- (APC-) H7 mouse anti-human CD4 and PerCP-Cy™5.5 mouse anti-human CD3 (BD Pharmingen, San Diego, CA, USA) for 30 min. After staining, the cells were fixed and permeabilized using the permeabilization/fixation solution kit (eBioscience, San Diego, USA) for 30 min, followed by staining with PE-Cy7-conjugated anti-IFN-γ (BD Biosciences, San Diego, CA, USA) for 30 min. .. The frequency of CD3+ CD4+ IFN-γ + (Th1) T cells was determined by flow cytometry analysis using the BD FACSAria II (Becton Dickinson, Washington, DC, USA) and analyzed with the FlowJo7.6.2 software (FlowJo LLC, Ashland, OR, USA).

Flow Cytometry:

Article Title: Gene expression profile of peripheral blood mononuclear cells in response to HIV-VLPs stimulation
Article Snippet: In parallel, PBMCs isolated by Ficoll-Hypaque density gradient centrifugation from same normal healthy donors, were pulsed with same concentration of HIV-VLPs for 12 hours. .. Analysis of DC phenotype MDDCs and PBMCs were incubated for 30 min at 4°C with murine monoclonal antibodies specific for CD80, CD83, CD86, HLA-DR and CD14 [PBMCs] (BD Pharmingen, San Diego, CA), washed and then fixed with 2% paraformaldehyde for analysis with a FACScalibur flow cytometer (BD Pharmingen). ..

Article Title: Dynamic Changes of Th1 Cytokines and the Clinical Significance of the IFN-γ/TNF-α Ratio in Acute Brucellosis
Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus density-gradient media (Amersham Biosciences, Little Chalfont, UK) according to the manufacturer's protocol. .. Flow Cytometry and Cytometric Bead Array The PBMCs were harvested and stained with allophycocyanin- (APC-) H7 mouse anti-human CD4 and PerCP-Cy™5.5 mouse anti-human CD3 (BD Pharmingen, San Diego, CA, USA) for 30 min. After staining, the cells were fixed and permeabilized using the permeabilization/fixation solution kit (eBioscience, San Diego, USA) for 30 min, followed by staining with PE-Cy7-conjugated anti-IFN-γ (BD Biosciences, San Diego, CA, USA) for 30 min. .. The frequency of CD3+ CD4+ IFN-γ + (Th1) T cells was determined by flow cytometry analysis using the BD FACSAria II (Becton Dickinson, Washington, DC, USA) and analyzed with the FlowJo7.6.2 software (FlowJo LLC, Ashland, OR, USA).

Cytometry:

Article Title: Gene expression profile of peripheral blood mononuclear cells in response to HIV-VLPs stimulation
Article Snippet: In parallel, PBMCs isolated by Ficoll-Hypaque density gradient centrifugation from same normal healthy donors, were pulsed with same concentration of HIV-VLPs for 12 hours. .. Analysis of DC phenotype MDDCs and PBMCs were incubated for 30 min at 4°C with murine monoclonal antibodies specific for CD80, CD83, CD86, HLA-DR and CD14 [PBMCs] (BD Pharmingen, San Diego, CA), washed and then fixed with 2% paraformaldehyde for analysis with a FACScalibur flow cytometer (BD Pharmingen). ..

Article Title: Dynamic Changes of Th1 Cytokines and the Clinical Significance of the IFN-γ/TNF-α Ratio in Acute Brucellosis
Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus density-gradient media (Amersham Biosciences, Little Chalfont, UK) according to the manufacturer's protocol. .. Flow Cytometry and Cytometric Bead Array The PBMCs were harvested and stained with allophycocyanin- (APC-) H7 mouse anti-human CD4 and PerCP-Cy™5.5 mouse anti-human CD3 (BD Pharmingen, San Diego, CA, USA) for 30 min. After staining, the cells were fixed and permeabilized using the permeabilization/fixation solution kit (eBioscience, San Diego, USA) for 30 min, followed by staining with PE-Cy7-conjugated anti-IFN-γ (BD Biosciences, San Diego, CA, USA) for 30 min. .. The frequency of CD3+ CD4+ IFN-γ + (Th1) T cells was determined by flow cytometry analysis using the BD FACSAria II (Becton Dickinson, Washington, DC, USA) and analyzed with the FlowJo7.6.2 software (FlowJo LLC, Ashland, OR, USA).

Isolation:

Article Title: Decreased Expression of TIM-3 on Th17 Cells Associated with Ophthalmopathy in Patients with Graves’ Disease
Article Snippet: .. 2.2 Isolation of Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells (PBMCs) were obtained from 2ml EDTA-k2 treated blood sample with 1 × BD Pharm Lyse lysing solution (BD Pharmingen, USA) according to the instructions from the manufacturer. .. PBMCs were washed twice in 10ml phosphate buffered saline (PBS) (Corning, Manassas, VA, USA) with 1% fetal bovine serum (FBS) (Gibco, Life Technologies, USA) and collected by centrifugation at the speed of 300× g for 5 min.

Article Title: Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves
Article Snippet: Temperatures of 35±2 °C are maintained on the microfluidic chip via a thermoelectric device on which the OCPW system is mounted, where steady-state temperatures are typically achieved on the order of seconds . .. Peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) are isolated from blood collected in EDTA vacutainer tubes (BD Biosciences, USA) from healthy volunteers at the Monash Precinct at the Alfred Hospital, Melbourne, Australia (Human Ethics Committee Approval 2007002182) via Ficoll density centrifugation (GE Healthcare, Sweden), and washed three times in Dulbecco's phosphate buffered saline (dPBS, Sigma-Aldrich, USA). .. PBMCs are then fluorescently labelled by incubation for 5 min at in 0.4 μM carboxyfluorescein succinimidyl ester in dPBS at 37 °C at 106 cells per ml, followed by quenching in ice-cold PBS media supplemented with 10% FCS (P10).

Marker:

Article Title: Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome
Article Snippet: .. Heparinized peripheral blood was incubated in the dark at room temperature for 15 min with Peridinin chlorophyll protein-conjugated anti-CD45 (a PBMC marker) Ab (Becton Dickinson) and either phycoerythrin-conjugated anti-MCP Ab (anti-CD46, clone 122-2, Serotec), or phycoerythrin-conjugated isotype control Ab (Serotec). ..

Centrifugation:

Article Title: Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves
Article Snippet: Temperatures of 35±2 °C are maintained on the microfluidic chip via a thermoelectric device on which the OCPW system is mounted, where steady-state temperatures are typically achieved on the order of seconds . .. Peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) are isolated from blood collected in EDTA vacutainer tubes (BD Biosciences, USA) from healthy volunteers at the Monash Precinct at the Alfred Hospital, Melbourne, Australia (Human Ethics Committee Approval 2007002182) via Ficoll density centrifugation (GE Healthcare, Sweden), and washed three times in Dulbecco's phosphate buffered saline (dPBS, Sigma-Aldrich, USA). .. PBMCs are then fluorescently labelled by incubation for 5 min at in 0.4 μM carboxyfluorescein succinimidyl ester in dPBS at 37 °C at 106 cells per ml, followed by quenching in ice-cold PBS media supplemented with 10% FCS (P10).

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    Flow <t>cytometric</t> analyses of interferon gamma (IFN-γ) expression on NK cells and NK cell subsets in <t>PBMCs</t> isolated from normal-weight (nw) and obese (ob) individuals. a , b Exemplary FACS plots of IFN-γ expression in CD56 bright and CD56 dim NK cells of a normal-weight and an obese individual. c Frequency of IFN-γ-expressing NK cells in PBMCs. d , e IFN-γ expression in CD56 bright ( d ) and CD56 dim ( e ) NK cells. Data are expressed as mean ± SEM. * P
    Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmcs/product/Becton Dickinson
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbmcs - by Bioz Stars, 2021-03
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    Becton Dickinson peripheral blood mononuclear cells pbmcs
    Irradiation-induced angiogenesis is dictated by MMP-9–mediated factor release from mast and stroma cells and incorporation of BM-derived cells. (A–C) HL ischemia was induced in Sl/Sl d and WBB6F1 +/+ mice, followed by 2 Gy IR or no IR ( n = 7). <t>PBMCs</t> were analyzed for the presence of CFU-Cs (A) and CFU-ECs (B). (C) Plasma VEGF was determined ( n = 7/group). (D) HL ischemia was induced in MMP-9 +/+ mice followed by 2 Gy IR. Mice were subdivided into groups receiving neutralizing <t>mAbs</t> against VEGFR-1, VEGFR-2 or a combination of both mAbs. (a) Blood flow was determined. Insert: Percentage of mice showing limb rescue after ligation. (b) Macroscopic pictures taken of ischemic and contralateral limb 7 d after ligation (*P
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/Becton Dickinson
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2021-03
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    Becton Dickinson pbmc aliquots
    CXCR3 and CCR6 expression in T cells of major depressive disorder (MDD) patients and non-depressed controls. (A) CXCR3-expressing T cells were identified by flow cytometric analysis of peripheral blood mononuclear cells from MDD patients and matched non-depressed controls (CTR). Displayed values are frequencies of CXCR3 + T cells expressed as a percentage of live CD3 + lymphocytes from a representative case–control pair. (B) Percentages of CXCR3-expressing total T cells, <t>CD4</t> + , and CD8 + T cells were quantified in our cohort ( n = 40). (C,D) Similar analyses were conducted for the surface expression of CCR6 on total T cells as well as on the CD4 + and CD8 + T cell subsets. (E) The CXCR3 ligands CXCL10 and CXCL11 were quantified in sera of MDD patients and matched controls using a cytometric bead array ( n = 38). (F) Surface CD3 MFI levels were measured by flow cytometric analysis of CD4 + and CD8 + T cells from MDD patients and matched controls ( n = 40). All graphs depict medians with interquartile ranges. For all comparisons, the Wilcoxon signed-rank test was used. SSC-A, side scatter-area; CXCR3, CXC-chemokine receptor type 3; CCR6, CC-chemokine receptor type 6; MFI, median fluorescence intensity.
    Pbmc Aliquots, supplied by Becton Dickinson, 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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    Signal scatter plots comparing the intensities of gene expressions on microarray chips within <t>Ficoll</t> and CPT-derived <t>PBMC</t> and their immune cell subsets for each healthy donor tested. The expression signals of Ficoll-derived cells were plotted against the expression signals of CPT-derived cells for each cell type and donor. A line of perfect correlation is indicated in all plots. Pearson’s correlation coefficients between each pair of samples are shown at the bottom right of each plot. The data showed that the pattern of gene expression was very similar between Ficoll and CPT-derived cell types, as indicated by the high correlation coefficients
    Pbmc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmc/product/Becton Dickinson
    Average 86 stars, based on 1 article reviews
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    Image Search Results


    Flow cytometric analyses of interferon gamma (IFN-γ) expression on NK cells and NK cell subsets in PBMCs isolated from normal-weight (nw) and obese (ob) individuals. a , b Exemplary FACS plots of IFN-γ expression in CD56 bright and CD56 dim NK cells of a normal-weight and an obese individual. c Frequency of IFN-γ-expressing NK cells in PBMCs. d , e IFN-γ expression in CD56 bright ( d ) and CD56 dim ( e ) NK cells. Data are expressed as mean ± SEM. * P

    Journal: Immunologic Research

    Article Title: Impaired natural killer cell subset phenotypes in human obesity

    doi: 10.1007/s12026-018-8989-4

    Figure Lengend Snippet: Flow cytometric analyses of interferon gamma (IFN-γ) expression on NK cells and NK cell subsets in PBMCs isolated from normal-weight (nw) and obese (ob) individuals. a , b Exemplary FACS plots of IFN-γ expression in CD56 bright and CD56 dim NK cells of a normal-weight and an obese individual. c Frequency of IFN-γ-expressing NK cells in PBMCs. d , e IFN-γ expression in CD56 bright ( d ) and CD56 dim ( e ) NK cells. Data are expressed as mean ± SEM. * P

    Article Snippet: For flow cytometric analyses, PBMCs were stained with the following antibodies (all from BD Biosciences, San Diego, USA, unless otherwise indicated): CD3 conjugated with phycoerythrin (PE)-Cy 7, CD4 conjugated with allophycocyanin (APC), CD 8 conjugated with PE, CD14 conjugated with fluorescein isothiocyanate (FITC), CD20 conjugated with APC-H7, CD56 conjugated with APC, CD253 (TRAIL; tumor necrosis factor-related apoptosis-inducing ligand) conjugated with PE, Ob-R (leptin receptor) conjugated with fluorescein (R & D Systems, Minneapolis, MN, USA), CD314 (NKG2D) conjugated with PE-CF594, CD25 conjugated with PE CF594, CD69 conjugated with FITC, and CD107a conjugated with PE.

    Techniques: Flow Cytometry, Expressing, Isolation, FACS

    Flow cytometric analyses of NK cells and NK cell subsets in PBMCs (peripheral blood mononuclear cells) isolated from normal-weight (nw) and obese (ob) individuals. a , b Exemplary FACS plots of CD56 bright and CD56 dim NK cells of a normal-weight and an obese subject. c Frequency of NK cells in PBMCs. d , e Expression of CD56 bright ( d ) and CD56 dim ( e ) NK cells. Data are expressed as mean ± SEM. * P

    Journal: Immunologic Research

    Article Title: Impaired natural killer cell subset phenotypes in human obesity

    doi: 10.1007/s12026-018-8989-4

    Figure Lengend Snippet: Flow cytometric analyses of NK cells and NK cell subsets in PBMCs (peripheral blood mononuclear cells) isolated from normal-weight (nw) and obese (ob) individuals. a , b Exemplary FACS plots of CD56 bright and CD56 dim NK cells of a normal-weight and an obese subject. c Frequency of NK cells in PBMCs. d , e Expression of CD56 bright ( d ) and CD56 dim ( e ) NK cells. Data are expressed as mean ± SEM. * P

    Article Snippet: For flow cytometric analyses, PBMCs were stained with the following antibodies (all from BD Biosciences, San Diego, USA, unless otherwise indicated): CD3 conjugated with phycoerythrin (PE)-Cy 7, CD4 conjugated with allophycocyanin (APC), CD 8 conjugated with PE, CD14 conjugated with fluorescein isothiocyanate (FITC), CD20 conjugated with APC-H7, CD56 conjugated with APC, CD253 (TRAIL; tumor necrosis factor-related apoptosis-inducing ligand) conjugated with PE, Ob-R (leptin receptor) conjugated with fluorescein (R & D Systems, Minneapolis, MN, USA), CD314 (NKG2D) conjugated with PE-CF594, CD25 conjugated with PE CF594, CD69 conjugated with FITC, and CD107a conjugated with PE.

    Techniques: Flow Cytometry, Isolation, FACS, Expressing

    Flow cytometric analyses of NKG2D receptor expression on NK cells and NK cell subsets in PBMCs isolated from normal-weight (nw) and obese (ob) individuals. a , b Exemplary FACS plots of NKG2D expression in CD56 bright and CD56 dim NK cells of a normal-weight and an obese subject. c Frequency of NKG2D-positive NK cells in PBMCs. d , e Expression of NKG2D in CD56 bright ( d ) and CD56 dim ( e ) NK cells. Data are expressed as mean ± SEM. * P

    Journal: Immunologic Research

    Article Title: Impaired natural killer cell subset phenotypes in human obesity

    doi: 10.1007/s12026-018-8989-4

    Figure Lengend Snippet: Flow cytometric analyses of NKG2D receptor expression on NK cells and NK cell subsets in PBMCs isolated from normal-weight (nw) and obese (ob) individuals. a , b Exemplary FACS plots of NKG2D expression in CD56 bright and CD56 dim NK cells of a normal-weight and an obese subject. c Frequency of NKG2D-positive NK cells in PBMCs. d , e Expression of NKG2D in CD56 bright ( d ) and CD56 dim ( e ) NK cells. Data are expressed as mean ± SEM. * P

    Article Snippet: For flow cytometric analyses, PBMCs were stained with the following antibodies (all from BD Biosciences, San Diego, USA, unless otherwise indicated): CD3 conjugated with phycoerythrin (PE)-Cy 7, CD4 conjugated with allophycocyanin (APC), CD 8 conjugated with PE, CD14 conjugated with fluorescein isothiocyanate (FITC), CD20 conjugated with APC-H7, CD56 conjugated with APC, CD253 (TRAIL; tumor necrosis factor-related apoptosis-inducing ligand) conjugated with PE, Ob-R (leptin receptor) conjugated with fluorescein (R & D Systems, Minneapolis, MN, USA), CD314 (NKG2D) conjugated with PE-CF594, CD25 conjugated with PE CF594, CD69 conjugated with FITC, and CD107a conjugated with PE.

    Techniques: Flow Cytometry, Expressing, Isolation, FACS

    Flow cytometric analyses of leptin receptor (Ob-R) expression in NK cells and NK cell subsets in PBMCs isolated from normal-weight (nw) and obese (ob) individuals. a Frequency of Ob-R expressing NK cells in PBMCs. b , c Expression of Ob-R in CD56 bright ( b ) and CD56 dim ( c ) NK cells. Data are expressed as mean ± SEM

    Journal: Immunologic Research

    Article Title: Impaired natural killer cell subset phenotypes in human obesity

    doi: 10.1007/s12026-018-8989-4

    Figure Lengend Snippet: Flow cytometric analyses of leptin receptor (Ob-R) expression in NK cells and NK cell subsets in PBMCs isolated from normal-weight (nw) and obese (ob) individuals. a Frequency of Ob-R expressing NK cells in PBMCs. b , c Expression of Ob-R in CD56 bright ( b ) and CD56 dim ( c ) NK cells. Data are expressed as mean ± SEM

    Article Snippet: For flow cytometric analyses, PBMCs were stained with the following antibodies (all from BD Biosciences, San Diego, USA, unless otherwise indicated): CD3 conjugated with phycoerythrin (PE)-Cy 7, CD4 conjugated with allophycocyanin (APC), CD 8 conjugated with PE, CD14 conjugated with fluorescein isothiocyanate (FITC), CD20 conjugated with APC-H7, CD56 conjugated with APC, CD253 (TRAIL; tumor necrosis factor-related apoptosis-inducing ligand) conjugated with PE, Ob-R (leptin receptor) conjugated with fluorescein (R & D Systems, Minneapolis, MN, USA), CD314 (NKG2D) conjugated with PE-CF594, CD25 conjugated with PE CF594, CD69 conjugated with FITC, and CD107a conjugated with PE.

    Techniques: Flow Cytometry, Expressing, Isolation

    Flow cytometric analyses of CD69 expression on NK cells and NK cell subsets in PBMCs isolated from normal-weight (nw) and obese (ob) individuals. a , b Exemplary FACS plots of CD69 expression in CD56 bright and CD56 dim NK cells of a normal-weight and an obese subject. c Frequency of CD69-positive NK cells in PBMCs. d , e Expression of CD69 in CD56 bright ( d ) and CD56 dim ( e ) NK cells. Data are expressed as mean ± SEM. * P

    Journal: Immunologic Research

    Article Title: Impaired natural killer cell subset phenotypes in human obesity

    doi: 10.1007/s12026-018-8989-4

    Figure Lengend Snippet: Flow cytometric analyses of CD69 expression on NK cells and NK cell subsets in PBMCs isolated from normal-weight (nw) and obese (ob) individuals. a , b Exemplary FACS plots of CD69 expression in CD56 bright and CD56 dim NK cells of a normal-weight and an obese subject. c Frequency of CD69-positive NK cells in PBMCs. d , e Expression of CD69 in CD56 bright ( d ) and CD56 dim ( e ) NK cells. Data are expressed as mean ± SEM. * P

    Article Snippet: For flow cytometric analyses, PBMCs were stained with the following antibodies (all from BD Biosciences, San Diego, USA, unless otherwise indicated): CD3 conjugated with phycoerythrin (PE)-Cy 7, CD4 conjugated with allophycocyanin (APC), CD 8 conjugated with PE, CD14 conjugated with fluorescein isothiocyanate (FITC), CD20 conjugated with APC-H7, CD56 conjugated with APC, CD253 (TRAIL; tumor necrosis factor-related apoptosis-inducing ligand) conjugated with PE, Ob-R (leptin receptor) conjugated with fluorescein (R & D Systems, Minneapolis, MN, USA), CD314 (NKG2D) conjugated with PE-CF594, CD25 conjugated with PE CF594, CD69 conjugated with FITC, and CD107a conjugated with PE.

    Techniques: Flow Cytometry, Expressing, Isolation, FACS

    Irradiation-induced angiogenesis is dictated by MMP-9–mediated factor release from mast and stroma cells and incorporation of BM-derived cells. (A–C) HL ischemia was induced in Sl/Sl d and WBB6F1 +/+ mice, followed by 2 Gy IR or no IR ( n = 7). PBMCs were analyzed for the presence of CFU-Cs (A) and CFU-ECs (B). (C) Plasma VEGF was determined ( n = 7/group). (D) HL ischemia was induced in MMP-9 +/+ mice followed by 2 Gy IR. Mice were subdivided into groups receiving neutralizing mAbs against VEGFR-1, VEGFR-2 or a combination of both mAbs. (a) Blood flow was determined. Insert: Percentage of mice showing limb rescue after ligation. (b) Macroscopic pictures taken of ischemic and contralateral limb 7 d after ligation (*P

    Journal: The Journal of Experimental Medicine

    Article Title: Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization

    doi: 10.1084/jem.20050959

    Figure Lengend Snippet: Irradiation-induced angiogenesis is dictated by MMP-9–mediated factor release from mast and stroma cells and incorporation of BM-derived cells. (A–C) HL ischemia was induced in Sl/Sl d and WBB6F1 +/+ mice, followed by 2 Gy IR or no IR ( n = 7). PBMCs were analyzed for the presence of CFU-Cs (A) and CFU-ECs (B). (C) Plasma VEGF was determined ( n = 7/group). (D) HL ischemia was induced in MMP-9 +/+ mice followed by 2 Gy IR. Mice were subdivided into groups receiving neutralizing mAbs against VEGFR-1, VEGFR-2 or a combination of both mAbs. (a) Blood flow was determined. Insert: Percentage of mice showing limb rescue after ligation. (b) Macroscopic pictures taken of ischemic and contralateral limb 7 d after ligation (*P

    Article Snippet: To understand which cell types increase after IR, we analyzed peripheral blood mononuclear cells (PBMCs) via FACS (Becton Dickinson) using mAbs against c-kit and VEGFR-2 after IR.

    Techniques: Irradiation, Derivative Assay, Mouse Assay, Flow Cytometry, Ligation

    CXCR3 and CCR6 expression in T cells of major depressive disorder (MDD) patients and non-depressed controls. (A) CXCR3-expressing T cells were identified by flow cytometric analysis of peripheral blood mononuclear cells from MDD patients and matched non-depressed controls (CTR). Displayed values are frequencies of CXCR3 + T cells expressed as a percentage of live CD3 + lymphocytes from a representative case–control pair. (B) Percentages of CXCR3-expressing total T cells, CD4 + , and CD8 + T cells were quantified in our cohort ( n = 40). (C,D) Similar analyses were conducted for the surface expression of CCR6 on total T cells as well as on the CD4 + and CD8 + T cell subsets. (E) The CXCR3 ligands CXCL10 and CXCL11 were quantified in sera of MDD patients and matched controls using a cytometric bead array ( n = 38). (F) Surface CD3 MFI levels were measured by flow cytometric analysis of CD4 + and CD8 + T cells from MDD patients and matched controls ( n = 40). All graphs depict medians with interquartile ranges. For all comparisons, the Wilcoxon signed-rank test was used. SSC-A, side scatter-area; CXCR3, CXC-chemokine receptor type 3; CCR6, CC-chemokine receptor type 6; MFI, median fluorescence intensity.

    Journal: Frontiers in Immunology

    Article Title: T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder

    doi: 10.3389/fimmu.2018.00291

    Figure Lengend Snippet: CXCR3 and CCR6 expression in T cells of major depressive disorder (MDD) patients and non-depressed controls. (A) CXCR3-expressing T cells were identified by flow cytometric analysis of peripheral blood mononuclear cells from MDD patients and matched non-depressed controls (CTR). Displayed values are frequencies of CXCR3 + T cells expressed as a percentage of live CD3 + lymphocytes from a representative case–control pair. (B) Percentages of CXCR3-expressing total T cells, CD4 + , and CD8 + T cells were quantified in our cohort ( n = 40). (C,D) Similar analyses were conducted for the surface expression of CCR6 on total T cells as well as on the CD4 + and CD8 + T cell subsets. (E) The CXCR3 ligands CXCL10 and CXCL11 were quantified in sera of MDD patients and matched controls using a cytometric bead array ( n = 38). (F) Surface CD3 MFI levels were measured by flow cytometric analysis of CD4 + and CD8 + T cells from MDD patients and matched controls ( n = 40). All graphs depict medians with interquartile ranges. For all comparisons, the Wilcoxon signed-rank test was used. SSC-A, side scatter-area; CXCR3, CXC-chemokine receptor type 3; CCR6, CC-chemokine receptor type 6; MFI, median fluorescence intensity.

    Article Snippet: Cell Purification For qRT-PCR and TCR sequencing analyses, CD4+ T cells were purified using magnetic beads (negative selection by BD IMag Human CD4 T Lymphocyte Enrichment Set, BD Biosciences) from PBMC aliquots thawed in cell separation buffer (1% human serum, 2 mM EDTA in PBS).

    Techniques: Expressing, Flow Cytometry, Fluorescence

    Surface and intracellular staining of CXCR3. (A) Percentages of CXCR3- and CCR6-expressing CD3 − lymphocytes (non-T cells) were quantified in major depressive disorder (MDD) patients and matched non-depressed controls (CTR) ( n = 40). (B) A representative plot shows fluorescence intensity of CXCR3 expression in intact (surface CXCR3; light gray-shaded curve) relative to fixed and permeabilized T cells (total cellular CXCR3; dark gray-shaded curve). Isotype-matched negative controls were used at the same concentration before fixation (black-dashed curve) and after fixation-permeabilization (gray-dashed curve) and showed no positive staining for CXCR3. (C) Total cellular CXCR3 MFI levels were measured by flow cytometric analysis of fixed and permeabilized peripheral blood mononuclear cells (PBMCs) from MDD patients and matched controls ( n = 36). Stained PBMCs were gated on live CD3 + lymphocytes (T cells), CD4 + and CD8 + T cell subsets as well as CD3 − lymphocytes (non-T cells). Graphs depict medians with interquartile ranges. For all comparisons, the Wilcoxon signed-rank test was used. CXCR3: CXC-chemokine receptor type 3; CCR6: CC-chemokine receptor type 6; MFI: median fluorescence intensity.

    Journal: Frontiers in Immunology

    Article Title: T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder

    doi: 10.3389/fimmu.2018.00291

    Figure Lengend Snippet: Surface and intracellular staining of CXCR3. (A) Percentages of CXCR3- and CCR6-expressing CD3 − lymphocytes (non-T cells) were quantified in major depressive disorder (MDD) patients and matched non-depressed controls (CTR) ( n = 40). (B) A representative plot shows fluorescence intensity of CXCR3 expression in intact (surface CXCR3; light gray-shaded curve) relative to fixed and permeabilized T cells (total cellular CXCR3; dark gray-shaded curve). Isotype-matched negative controls were used at the same concentration before fixation (black-dashed curve) and after fixation-permeabilization (gray-dashed curve) and showed no positive staining for CXCR3. (C) Total cellular CXCR3 MFI levels were measured by flow cytometric analysis of fixed and permeabilized peripheral blood mononuclear cells (PBMCs) from MDD patients and matched controls ( n = 36). Stained PBMCs were gated on live CD3 + lymphocytes (T cells), CD4 + and CD8 + T cell subsets as well as CD3 − lymphocytes (non-T cells). Graphs depict medians with interquartile ranges. For all comparisons, the Wilcoxon signed-rank test was used. CXCR3: CXC-chemokine receptor type 3; CCR6: CC-chemokine receptor type 6; MFI: median fluorescence intensity.

    Article Snippet: Cell Purification For qRT-PCR and TCR sequencing analyses, CD4+ T cells were purified using magnetic beads (negative selection by BD IMag Human CD4 T Lymphocyte Enrichment Set, BD Biosciences) from PBMC aliquots thawed in cell separation buffer (1% human serum, 2 mM EDTA in PBS).

    Techniques: Staining, Expressing, Fluorescence, Concentration Assay, Flow Cytometry

    Regulatory T cells in major depressive disorder (MDD) patients and non-depressed controls. (A) Regulatory T cells (Tregs) were identified by flow cytometric analysis of peripheral blood mononuclear cells from MDD patients and matched non-depressed controls (CTR). Displayed values are frequencies of Tregs expressed as a percentage of live CD4 + T cells from a representative case–control pair. (B) Differences in Treg frequency are depicted for the entire cohort ( n = 40). (C) Negatively selected CD4 + T cells from a subsample of patients and matched controls ( n = 20) were analyzed for mRNA expression of the T helper-associated transcription factors Forkhead box P3 ( FOXP3 ), T-box 21 ( T-bet ), GATA binding protein 3 ( GATA3 ), and RAR related orphan receptor C ( RORC ), respectively. Expression was normalized to the geometric mean expression of three housekeeping genes ( IPO8, TBP, RPL13A ). (D) The correlation between the expression levels of the gene FOXP3 in purified CD4 + T cells and the frequency of Tregs expressed as a percentage of CD4 + T cells is plotted ( n = 20). Graphs depict medians with interquartile ranges. For all comparisons, the Wilcoxon signed-rank test was used.

    Journal: Frontiers in Immunology

    Article Title: T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder

    doi: 10.3389/fimmu.2018.00291

    Figure Lengend Snippet: Regulatory T cells in major depressive disorder (MDD) patients and non-depressed controls. (A) Regulatory T cells (Tregs) were identified by flow cytometric analysis of peripheral blood mononuclear cells from MDD patients and matched non-depressed controls (CTR). Displayed values are frequencies of Tregs expressed as a percentage of live CD4 + T cells from a representative case–control pair. (B) Differences in Treg frequency are depicted for the entire cohort ( n = 40). (C) Negatively selected CD4 + T cells from a subsample of patients and matched controls ( n = 20) were analyzed for mRNA expression of the T helper-associated transcription factors Forkhead box P3 ( FOXP3 ), T-box 21 ( T-bet ), GATA binding protein 3 ( GATA3 ), and RAR related orphan receptor C ( RORC ), respectively. Expression was normalized to the geometric mean expression of three housekeeping genes ( IPO8, TBP, RPL13A ). (D) The correlation between the expression levels of the gene FOXP3 in purified CD4 + T cells and the frequency of Tregs expressed as a percentage of CD4 + T cells is plotted ( n = 20). Graphs depict medians with interquartile ranges. For all comparisons, the Wilcoxon signed-rank test was used.

    Article Snippet: Cell Purification For qRT-PCR and TCR sequencing analyses, CD4+ T cells were purified using magnetic beads (negative selection by BD IMag Human CD4 T Lymphocyte Enrichment Set, BD Biosciences) from PBMC aliquots thawed in cell separation buffer (1% human serum, 2 mM EDTA in PBS).

    Techniques: Flow Cytometry, Expressing, Binding Assay, Purification

    Peripheral blood counts and frequencies of major leukocyte subsets. (A) Absolute peripheral blood granulocyte, monocyte, and lymphocyte counts were obtained from major depressive disorder (MDD) patients and matched non-depressed controls (CTR) using a Coulter Ac·T Diff hematology analyzer ( n = 40). (B) Frequencies of total CD3 + lymphocytes (T cells), CD3 − CD56 − CD19 + B cells and CD3 − CD19 − CD20 − CD14 − CD56 + natural killer (NK) cells were obtained by flow cytometric analysis of thawed peripheral blood mononuclear cells. (C) T cells were further discriminated into CD4 + CD8 − and CD8 + CD4 − subsets. (D) Among NK cells, CD56 low CD16 + cytotoxic (NKc) cells and CD56 high CD16 − regulatory (NKreg) cells were also identified. Graphs depict medians with interquartile ranges. For all comparisons, the Wilcoxon signed-rank test was used.

    Journal: Frontiers in Immunology

    Article Title: T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder

    doi: 10.3389/fimmu.2018.00291

    Figure Lengend Snippet: Peripheral blood counts and frequencies of major leukocyte subsets. (A) Absolute peripheral blood granulocyte, monocyte, and lymphocyte counts were obtained from major depressive disorder (MDD) patients and matched non-depressed controls (CTR) using a Coulter Ac·T Diff hematology analyzer ( n = 40). (B) Frequencies of total CD3 + lymphocytes (T cells), CD3 − CD56 − CD19 + B cells and CD3 − CD19 − CD20 − CD14 − CD56 + natural killer (NK) cells were obtained by flow cytometric analysis of thawed peripheral blood mononuclear cells. (C) T cells were further discriminated into CD4 + CD8 − and CD8 + CD4 − subsets. (D) Among NK cells, CD56 low CD16 + cytotoxic (NKc) cells and CD56 high CD16 − regulatory (NKreg) cells were also identified. Graphs depict medians with interquartile ranges. For all comparisons, the Wilcoxon signed-rank test was used.

    Article Snippet: Cell Purification For qRT-PCR and TCR sequencing analyses, CD4+ T cells were purified using magnetic beads (negative selection by BD IMag Human CD4 T Lymphocyte Enrichment Set, BD Biosciences) from PBMC aliquots thawed in cell separation buffer (1% human serum, 2 mM EDTA in PBS).

    Techniques: Flow Cytometry

    Signal scatter plots comparing the intensities of gene expressions on microarray chips within Ficoll and CPT-derived PBMC and their immune cell subsets for each healthy donor tested. The expression signals of Ficoll-derived cells were plotted against the expression signals of CPT-derived cells for each cell type and donor. A line of perfect correlation is indicated in all plots. Pearson’s correlation coefficients between each pair of samples are shown at the bottom right of each plot. The data showed that the pattern of gene expression was very similar between Ficoll and CPT-derived cell types, as indicated by the high correlation coefficients

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Signal scatter plots comparing the intensities of gene expressions on microarray chips within Ficoll and CPT-derived PBMC and their immune cell subsets for each healthy donor tested. The expression signals of Ficoll-derived cells were plotted against the expression signals of CPT-derived cells for each cell type and donor. A line of perfect correlation is indicated in all plots. Pearson’s correlation coefficients between each pair of samples are shown at the bottom right of each plot. The data showed that the pattern of gene expression was very similar between Ficoll and CPT-derived cell types, as indicated by the high correlation coefficients

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Microarray, Cycling Probe Technology, Derivative Assay, Expressing

    Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology

    Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Expressing, Derivative Assay, Isolation, Cycling Probe Technology, Microarray

    Yield and quality of RNA recovered from total PBMC and their individual immune cell subsets did not significantly differ between PBMC isolated by Ficoll and CPT methods. a The mean amount of RNA per cell and ( b ) the mean RIN of RNA preparations extracted from total PBMC and their individual immune cell subsets derived from PBMC prepared by Ficoll and CPT methods from 6 health donors. There were no significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Yield and quality of RNA recovered from total PBMC and their individual immune cell subsets did not significantly differ between PBMC isolated by Ficoll and CPT methods. a The mean amount of RNA per cell and ( b ) the mean RIN of RNA preparations extracted from total PBMC and their individual immune cell subsets derived from PBMC prepared by Ficoll and CPT methods from 6 health donors. There were no significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology, Derivative Assay

    Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and ( b ) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and ( b ) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Cycling Probe Technology, Isolation

    Schematic outline of the study. PBMC from 6 healthy donors were isolated using Ficoll-Paque gradient fractionation or BD Vacutainer CPT protocol, and cell yield and purity were compared. Subsequently, immune cell subsets were separated by positive selection from PBMC obtained by both methods, and yields and viabilities of the subsets were determined and compared. RNA and DNA were extracted from total PBMC isolated by both protocols and from their subsets, and the nucleic acid yield and quality compared. Finally, gene expression analysis of individual immune cell subsets and total PBMC obtained by Ficoll and CPT isolation methods were performed and the results compared. LN 2 , liquid nitrogen

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Schematic outline of the study. PBMC from 6 healthy donors were isolated using Ficoll-Paque gradient fractionation or BD Vacutainer CPT protocol, and cell yield and purity were compared. Subsequently, immune cell subsets were separated by positive selection from PBMC obtained by both methods, and yields and viabilities of the subsets were determined and compared. RNA and DNA were extracted from total PBMC isolated by both protocols and from their subsets, and the nucleic acid yield and quality compared. Finally, gene expression analysis of individual immune cell subsets and total PBMC obtained by Ficoll and CPT isolation methods were performed and the results compared. LN 2 , liquid nitrogen

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Fractionation, Cycling Probe Technology, Selection, Expressing

    Yield and viability of PBMC isolated by the Ficoll and CPT protocols. a The mean numbers of PBMC per ml of blood obtained by Ficoll or CPT isolation procedure from 6 healthy donors. b The mean viability of PBMC freshly isolated from the same 6 healthy donors by either Ficoll gradient separation or CPT technique. No significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Yield and viability of PBMC isolated by the Ficoll and CPT protocols. a The mean numbers of PBMC per ml of blood obtained by Ficoll or CPT isolation procedure from 6 healthy donors. b The mean viability of PBMC freshly isolated from the same 6 healthy donors by either Ficoll gradient separation or CPT technique. No significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology

    Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology, Flow Cytometry, Cytometry

    Post-cryopreservation recovery and viability of PBMC isolated by the Ficoll and CPT methods. a The mean percent recovery after cryopreservation of PBMC collected using the Ficoll and CPT protocols. Percent recovery was calculated by dividing the number of PBMC recovered after thawing by the number of cells that were cryopreserved. b The mean viability of recovered PBMC isolated by the Ficoll and CPT technique. No significant differences ( P

    Journal: BMC Immunology

    Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

    doi: 10.1186/s12865-015-0113-0

    Figure Lengend Snippet: Post-cryopreservation recovery and viability of PBMC isolated by the Ficoll and CPT methods. a The mean percent recovery after cryopreservation of PBMC collected using the Ficoll and CPT protocols. Percent recovery was calculated by dividing the number of PBMC recovered after thawing by the number of cells that were cryopreserved. b The mean viability of recovered PBMC isolated by the Ficoll and CPT technique. No significant differences ( P

    Article Snippet: About 45 ml of blood was obtained from each donor using BD Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which PBMC were isolated by Ficoll-Paque gradient centrifugation (see below).

    Techniques: Isolation, Cycling Probe Technology