Journal: Physiological Reports

Article Title: Cyclophosphamide treatment for hypertension and renal injury in an experimental model of systemic lupus erythematosus

doi: 10.14814/phy2.14059

Figure Lengend Snippet: Impact of cyclophosphamide (CYC) on circulating immune cells. Flow cytometry was performed on cryopreserved peripheral blood leukocytes to determine the impact of CYC treatment on lymphoid and myeloid cell populations. (A) Representative scatter plot of peripheral blood leukocytes. (B) Data suggest that CYC treatment reduced CD45R + B cells compared to vehicle treatment in systemic lupus erythematosus (SLE) mice (15.87 ± 8.49% vs. 26.96 ± 4.72%, P = 0.06). (C) There was no significant difference in the percentage of circulating CD3 + CD4 + T cells between groups or (D) the percentage of CD3 + CD8 + T cells.(E) The percentage of circulating neutrophils was significantly increased in CYC‐treated SLE mice compared to vehicle‐treated mice (39.26 ± 4.92 vs. 20.58 ± 6.01, * P < 0.05). (F) There was no significant different in the percentage of circulating monocytes between groups. ○ SLE Vehicle ( n = 8), and ● SLE CYC ( n = 10).

Article Snippet: The following antibodies were diluted with wash buffer (50 μL/sample; 1:100 dilution), and placed in a single tube‐containing sample to measure the relative percentages of circulating lymphocyte populations: CD3e‐PE‐Cy7 (clone 145‐2C11), CD4‐FITC (clone GK1.5), CD8‐PerCP‐Cy (clone 53‐6.7), and CD45R‐Alexa Fluor (clone RA3‐6B2) (BD Biosciences).

Techniques: Flow Cytometry

Journal: Physiological Reports

Article Title: Cyclophosphamide treatment for hypertension and renal injury in an experimental model of systemic lupus erythematosus

doi: 10.14814/phy2.14059

Figure Lengend Snippet: Impact of cyclophosphamide (CYC) on renal lymphocyte infiltration. (A) Renal CD45R + B cells were significantly increased in systemic lupus erythematosus (SLE) vehicle‐treated mice compared to all other treatment groups (* P < 0.05) (B) Renal CD3 + CD4 + T cells were significantly increased in SLE vehicle‐treated mice compared to all other treatment groups (* P < 0.05). (C) Renal CD3 + CD8 + T cells were not significantly different in response to CYC treatment in control or SLE mice. □ Control Vehicle ( n = 5) ■ Control CYC ( n = 5), ○ SLE Vehicle ( n = 5), and ● SLE CYC ( n = 5).

Article Snippet: The following antibodies were diluted with wash buffer (50 μL/sample; 1:100 dilution), and placed in a single tube‐containing sample to measure the relative percentages of circulating lymphocyte populations: CD3e‐PE‐Cy7 (clone 145‐2C11), CD4‐FITC (clone GK1.5), CD8‐PerCP‐Cy (clone 53‐6.7), and CD45R‐Alexa Fluor (clone RA3‐6B2) (BD Biosciences).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Immune Cell Subsets in Peripheral Blood from Patients with Engineered Stone Silica-Induced Lung Inflammation

doi: 10.3390/ijms25115722

Figure Lengend Snippet: Total T cells and CD4 + , CD8 + subsets quantified by flow cytometry in peripheral blood from patients diagnosed with simple silicosis (SS) or pulmonary massive fibrosis (PMF) and healthy controls (HC). ( A ) Percentage of total T cells (CD3 + ). ( B ) Percentage of T helper cells (CD3 + CD4 + ). ( C ) Percentage of cytotoxic T cells (CD3 + CD8 + ). ( D ) CD4 + /CD8 + ratio. ° indicate outliers. * indicate significance at p < 0.05.

Article Snippet: Surface immunostaining of the cell population was performed by incubating 150 μL of whole peripheral blood for 15 min in the dark with the following antibodies in different combinations ( ): CD45-V500, CD3-APC-H7, CD4-V450, CD56-APC, CD19-PerCP-CY7, CD27-PE, CD16-FITC, CD45RA-FITC, and CD45RO-PE from BD (Becton Dickinson; San Jose, CA, USA); CD19-APC, CD38-FITC, and CD8-PerCP from Immunotools (Friesoythe, Germany); and CD127-PerCP-CY5.5 from BioLegend (San Diego, CA, USA).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Immune Cell Subsets in Peripheral Blood from Patients with Engineered Stone Silica-Induced Lung Inflammation

doi: 10.3390/ijms25115722

Figure Lengend Snippet: Analysis of the surface markers of the lymphocyte subsets.

Article Snippet: Surface immunostaining of the cell population was performed by incubating 150 μL of whole peripheral blood for 15 min in the dark with the following antibodies in different combinations ( ): CD45-V500, CD3-APC-H7, CD4-V450, CD56-APC, CD19-PerCP-CY7, CD27-PE, CD16-FITC, CD45RA-FITC, and CD45RO-PE from BD (Becton Dickinson; San Jose, CA, USA); CD19-APC, CD38-FITC, and CD8-PerCP from Immunotools (Friesoythe, Germany); and CD127-PerCP-CY5.5 from BioLegend (San Diego, CA, USA).

Techniques:

Journal: Nature Communications

Article Title: A child with perinatal HIV infection and long-term sustained virological control following antiretroviral treatment cessation

doi: 10.1038/s41467-019-08311-0

Figure Lengend Snippet: Detection of HIV RNA, HIV DNA and replication-competent virus. a Viral load results at 9.5 years of age when testing the standard 1 mL of plasma (target not detected (TND); left) vs. 10 mL (middle) by the standard Roche assay. A qualitative ultrasensitive RNA nested integrase PCR (IN-qPCR) assay conducted on 3 mL plasma; DNAse treated to ensure no contaminating cell-associated HIV-1 DNA (right). b Quantitation of the total HIV-1 DNA reservoir using a semi-nested quantitative reverse transcription PCR (RT-qPCR) assay at 9.5 years. The standard curve (orange squares) shows plasmid copy number controls (1–100,000 copies) on the x -axis and corresponding cycle threshold values on the y -axis. The case replicates are shown as blue squares. Curves lower than the 10 0 (1 copy) plasmid control are counted as 1 copy. c A neighbour-joining phylogenetic tree constructed using the partial gag-PR sequence (1414 bp HXB2 nt 903–2334; Gag aa39-501, PR aa 1–28). Reference subtypes A–D (in blue, black, purple and green, respectively) are included and the tree is rooted on SIV chimpanzee sequence (Los Alamos HIV sequence database; https://www.hiv.lanl.gov ). Accession numbers (e.g. AF067155) of reference sequences are indicated in the figure. Numbers at the nodes indicate percentage bootstrap scores ( n = 1000). The child’s (Case) gag consensus sequence (see Supplementary Fig. ) is indicated and clusters with subtype C sequences (purple). d The ability to reactivate virus from the child’s CD4+ T cells was measured using two co-culture methods: donor CD8-depleted peripheral blood mononuclear cells (PBMCs) and MOLT4/CCR5 cells (top of panel). Included is the healthy HIV-1-uninfected donor (negative control) and an HIV-1-infected patient with high viral load, CD4+ T cell count <200 cells per µL (positive control). The ability to infect CD4+ cells from the case with HIV-1 BaL (bottom of panel). Donor and MOLT4/CCR5 cells were included as positive controls. The case was tested at the indicated times (age). The different sizes and shades of blue colour of the circles represent the p24 concentration in culture supernatants; the actual pg mL −1 values appear within the circles (the colour key shows ranges of levels of p24 according to shade of blue, with p24 levels increasing with increasing intensity of colour). *Indicates the time point at which a very weak signal was obtained by ultrasensitive nested RNA IN-qPCR assay in the 50-week sample

Article Snippet: The following antibodies were used, in three antibody panels: CD3 APC-H7 (clone SK7, Cat. No. 560176, 2 µL), CD8 PerCP (clone SK1, Cat. No. 347314, 6.5 µL), CD8 Alexa Fluor 700 (clone RPA-T8, Cat. No. 557945, 2 µL), CD4 BV786 (clone L200, Cat. No. 563914, 0.8 µL), CD4 FITC (clone SK3, Cat. No. 347413, 6 µL), CCR5 PE (clone 2D7, custom 1:1 conjugated antibody, 10 µL), CCR7 FITC (clone 150503, Cat. No. 150503, 6.5 µL), CD45RO BV510 (clone UCHL1, Cat. No. 563215, 4 µL), CD62L PE-CF594 (clone DREG-56, 1.6 µL), PD-1 BV786 (clone EH12.1, 3.3 µL) were obtained from BD Biosciences.

Techniques: Quantitation Assay, Quantitative RT-PCR, Plasmid Preparation, Construct, Sequencing, Co-Culture Assay, Negative Control, Infection, Cell Counting, Positive Control, Concentration Assay

Journal: BioTechnologia

Article Title: In vitro immune evaluation of adenoviral vector-based platform for infectious diseases

doi: 10.5114/bta.2023.132775

Figure Lengend Snippet: The percentage of the T lymphocyte subpopulations among PBMCs stimulated with the vaccine factors ( n = 6) compared with PBMC mock (PBMCs stimulated with LPS at 1.25 μg/ml) after the 24-h treatment at 37°C in 5% CO 2 ; (A) the percentage of CD4 + and CD8 + T among CD3 + ; (B) the percentage of central memory (CM), effector memory (EM), effector memory terminally differentiated (EMRA), and CD197 + CD45RA + among CD4 + ; (C) the heat map analysis of the markers expressed by the subpopulations of CD4 + CD45RA + CD197 + ; the expression of CD95 (T SCM ) after the treatment with AdV1 (left top row) or rRBD+Ab (left bottom row); the expression of NAÏVE (CD95 - ) after the treatment with rRBD (right bottom row); (D) CM, EM, EMRA, and CD45RA + CD197 + among CD8 + ; (E) The heat map analysis of markers expressed by the subpopulations of CD4 + CD45RA + CD197 + ; the expression of CD95 (T SCM ) after the treatment with AdV1 (left top row) or rRBD+Ab (left bottom row); the data are representative of 2–5 different experiments; multiple unpaired t -tests: only comparison with a P -value less than or equal to 0.05 was presented (significant diff. between the means of mock vs. the marked treatments, P ≤ 0.05)

Article Snippet: In brief, 50 μl of PBMCs (1 × 10 6 cells/ml) were incubated at room temperature in the dark for 30 min with the following antibodies: CD3 (APC-H7, cat. no. 560176), CD8 (PerCP, cat. no. 345774), CD4 (PE-Cy7, cat. no. 557852), CD197 (BB515, cat. no. 566764), CD45RA (APC, cat. no. 550855), and CD95 (PE, cat. no. 555674) (all from BD Biosciences USA).

Techniques: Expressing, Comparison