round-bottom polystyrene tubes, (flow cytometry tubes)  (Corning Life Sciences)

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: An Inflamed Human Alveolar Model for Testing the Efficiency of Anti-inflammatory Drugs in vitro

doi: 10.3389/fbioe.2020.00987

Figure Lengend Snippet: Phenotype shift in macrophage monocultures upon exposure to pro- and anti-inflammatory stimuli. (A) Secretion of pro-inflammatory mediators (IL-8 and TNF-α; in pg/mL) in cell-culture medium in the basal compartment, assessed via ELISA. (B) Expression of CD206 surface marker (median fluorescence intensity), denoting M2 phenotype, assessed via flow cytometry analysis. The CD206 intensity histogram is shown in . (C) Cell viability of MDMs after all of the treatments, assessed via membrane rupture (LDH assay), presented as fold increase over untreated cells (the dotted line). The data is presented as the mean of the four biological repetitions ± standard deviation, whereas individual values from biological repetitions are presented as circles, color-coded for the data from the same biological repetitions (donors). Statistically significant differences among the groups (One-way ANOVA, Tukey’s post hoc ; α = 0.05): * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001. Abbreviations: IL-8, interleukin 8; TNF-α, tumor necrosis factor α; MFI, median fluorescence intensity; CD206, cluster of differentiation 206, mannose receptor; IL-4+IL-13, interleukins 4 and 13 (both applied at 20 ng/mL for 48 h); LDH, lactate dehydrogenase; Pos. ctrl Triton X, positive control for LDH assay, i.e., cells exposed to 0.2% Triton X-100 (v/v; 24 h).

Article Snippet: Cells from two identically treated wells in a 6-well plate were pooled in flow cytometry tubes (FALCON ® 5 mL Polystyrene Round-Bottom Tube, Corning, Switzerland), counted, centrifuged (5702R, Eppendorf; 500 RCF, 5 min), and resuspended in cold flow cytometry buffer (PBS with 1% BSA, 0.1% NaN3, 1 mM ethylenediaminetetraacetic acid [EDTA] at pH 7.4).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Marker, Fluorescence, Flow Cytometry, Membrane, Lactate Dehydrogenase Assay, Standard Deviation, Positive Control

Journal: Frontiers in Immunology

Article Title: Type I Interferon Regulates the Survival and Functionality of B Cells in Rainbow Trout

doi: 10.3389/fimmu.2020.01494

Figure Lengend Snippet: Survival of blood IgM + IgD + B cells in response to type I and type II IFNs. PBLs were stimulated with 50 ng/ml rIFNa, 20 ng/ml rIFNγ or media alone (control) and cultured at 20°C for 72 h. Leukocytes were then labeled with specific monoclonal antibodies against trout IgM and IgD and analyzed by flow cytometry. Cells were gated on the basis of their FSC and SSC and percentages of IgM + IgD + cells determined on singlet and live (DAPI negative) cells. Representative dot plots from one individual fish are shown (A) along with mean percentages and total number of cells detected for IgM + IgD + B cells (B) and IgM − IgD − cells (C) (mean + SEM; n = 9). In an independent experiment, B cells were sorted from blood leukocytes using a biotinyilated Fab fragment of anti-IgM 1.14 and then incubated with the rIFNs as described above. After 72 h, the percentage of live IgM + IgD + B cells and the total number of live IgM + IgD + B cells determined by flow cytometry as described in the Materials and Methods section (mean + SEM; n = 7) (D) . Asterisks denote significant differences between samples treated with rIFNs and control samples (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001).

Article Snippet: The cells were then incubated for 15 min at room temperature (RT) in the dark before being transferred to flow cytometry tubes (Falcon® 5 mL Round Bottom Polystyrene Test Tube, Corning) containing 400 μl of 1X Annex V Binding Buffer, and analyzed within 1 h on a FACS Celesta flow cytometer.

Techniques: Control, Cell Culture, Labeling, Bioprocessing, Flow Cytometry, Incubation

Journal: Frontiers in Immunology

Article Title: Type I Interferon Regulates the Survival and Functionality of B Cells in Rainbow Trout

doi: 10.3389/fimmu.2020.01494

Figure Lengend Snippet: Effect of type I and type II IFNs on the antigen-presenting capacities of blood cells. PBLs were incubated with 50 ng/ml rIFNa, 20 ng/ml rIFNγ or media alone (control) for 72 h at 20°C. The level of MHC-II surface expression on IgM + IgD + B cells and IgM − IgD − cells was then measured via flow cytometry using a specific mAb against trout MHC-II. A representative histogram is shown together with a graph of the mean MHC-II MFI values for IgM + IgD + B cells (A) or IgM − IgD − cells (B) (mean + SEM; n = 9). In another experiment, blood leukocytes were treated with the rIFNs as described above and after 24 h IgM + IgD + B cells were sorted by flow cytometry. RNA was extracted to determine the levels of transcription of mhcII, cd83 , and cd80/86 (C) by real time PCR. Gene expression data were normalized against the endogenous control b-actin and are shown as relative expression (mean + SEM; n = 10). Asterisks denote significant differences between samples treated with rIFNs and control samples (* P ≤ 0.05 and ** P ≤ 0.01).

Article Snippet: The cells were then incubated for 15 min at room temperature (RT) in the dark before being transferred to flow cytometry tubes (Falcon® 5 mL Round Bottom Polystyrene Test Tube, Corning) containing 400 μl of 1X Annex V Binding Buffer, and analyzed within 1 h on a FACS Celesta flow cytometer.

Techniques: Incubation, Control, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Frontiers in Immunology

Article Title: Type I Interferon Regulates the Survival and Functionality of B Cells in Rainbow Trout

doi: 10.3389/fimmu.2020.01494

Figure Lengend Snippet: Induction of mx transcription in blood IgM + IgD + B cells in response to type I or type II IFNs. Blood leukocytes were incubated with 50 ng/ml rIFNa, 20 ng/ml rIFNγ or media alone (control) at 20°C. After 24 h, IgM + IgD + B cells were sorted by flow cytometry and RNA extracted to determine the levels of transcription of the different mx genes present in rainbow trout ( mx1 - mx9 ). Gene expression data were normalized against the endogenous control b-actin and are shown as relative expression (mean + SEM; n = 10). Asterisks denote significant differences between samples treated with rIFNs and control samples (* P ≤ 0.05 and ** P ≤ 0.01).

Article Snippet: The cells were then incubated for 15 min at room temperature (RT) in the dark before being transferred to flow cytometry tubes (Falcon® 5 mL Round Bottom Polystyrene Test Tube, Corning) containing 400 μl of 1X Annex V Binding Buffer, and analyzed within 1 h on a FACS Celesta flow cytometer.

Techniques: Incubation, Control, Flow Cytometry, Gene Expression, Expressing