cell surface marker (cd86)  (Thermo Fisher)

Journal: EMBO Reports

Article Title: Tissue‐like environments shape functional interactions of HIV ‐1 with immature dendritic cells

doi: 10.15252/embr.202356818

Figure Lengend Snippet: Monocytes isolated from PBMCs of healthy donors were differentiated into iDCs for 6 days by the addition of IL‐4/GM‐CSF, and the presence of maturation markers was analyzed by FACS and compared to LPS‐treated control to verify that DCs were in an immature state. A Representative histograms of FACS analysis showing fluorescence intensity for each marker analyzed. B Representative spinning disc microscopy images of iDC cultured in 2D suspension (left) or 3D collagen (right) at 4 days post‐seeding. Cells were stained with phalloidin‐Atto390 to visualize F‐actin and cell morphology. Scale bar = 50 μm. C Representative spinning disc microscopy images of iDC cultured in 2D suspension (left) or 3D collagen (right) at 4 h post‐seeding. Cells were stained with Wga‐Alexa 647 to visualize the plasma membrane and cell morphology. Scale bar = 20 μm. D The graphs show the quantifications of cell area, sphericity, and volume, in 2D suspension and 3D collagen, obtained using Imaris's surface finder option, based on Wga‐Alexa 647 staining. Lines are set at median values and each dot represents an analyzed cell. *** P < 0.001 calculated with Mann–Whitney test. E–G Comparison of deformation (E), volume (F), and Young's modulus (G) of iDCs derived from four donors ( n = 1,145 to n = 2,761 each, mean ± SD) cultured either in 2D suspension or 3D collagen for 2 days and measured with RT‐DC at a flow rate of 0.04 μl/s. For statistical significance testing, linear mixed‐model analysis was performed to calculate ANOVA P ‐values; * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available online for this figure.

Article Snippet: The following antibodies for the detection of cell surface markers were used at the indicated dilution: CD86 ANTIBODY, anti‐human, FITC, REAfinityTM (Miltenyi Biotec 130‐116‐262 1:100); CD86 antibody, anti‐human, PerCP‐Vio® 700, REAfinityTM (Miltenyi Biotec 130‐116‐164 1:100); CD80 antibody, anti‐human, PE (Miltenyi Biotec 130‐117‐683 1:100); PE mouse anti‐human CD83 (BD PharmigenTM 556855 1:20); PE/cyanine7 anti‐human CD1a antibody (Biolegend 300121 1:100); CD169 (Siglec‐1) antibody, anti‐human, PerCP‐Vio® 700, REAfinityTM (Miltenyi Biotec 130‐101‐508 1:100); BV421 anti‐human CD169 (Biolegend 346018 1:100); BV421 mouse anti‐human CD206 (BD Pharmigen 564062 1:20); FITC ##man CD209 (DC‐SIGN; BD PharmingenTM 551264 1:100); PerCP/cyanine5.5 anti‐human CD1c antibody (Biolegend 331514 1:50); Brilliant Violet 421TM anti‐human CD141 (Thrombomodulin) antibody (Biolegend 344113 1:50); CD327 (Siglec‐6) Antibody, anti‐human, FITC, REAfinityTM (Miltenyi Biotec 130‐112‐898 1:50); BV605 mouse anti‐human CD11c (Biolegend 301636 1:50); FITC anti‐human CD195 (CCR5) Antibody (Biolegend 359120 1:100); and PerCP/cyanine5.5 anti‐human CD4 Antibody (Biolegend 300530 1:100).

Techniques: Isolation, Control, Fluorescence, Marker, Microscopy, Cell Culture, Suspension, Staining, Clinical Proteomics, Membrane, MANN-WHITNEY, Comparison, Derivative Assay

Journal: EMBO Reports

Article Title: Tissue‐like environments shape functional interactions of HIV ‐1 with immature dendritic cells

doi: 10.15252/embr.202356818

Figure Lengend Snippet: Representative spinning disc confocal microscopy images of HIV‐1 NLENG R5 Vpr‐mRuby2 particles stained with anti‐HIV‐1 p24 antibody. Vpr‐mRuby2 is shown in red, while p24 is shown in green. Scale bar = 5 μm. Relative infectivity of NLENG‐1 R5 particles in the presence or absence of Vpr‐mRuby2 produced in 293T cells. Particle infectivity was measured by infecting TZM‐bl reporter cells. The number of blue cells present was normalized to the RT activity of the respective virus stock determined by SG‐PERT assay. Bars represent mean ± SD of four technical replicates. Representative spinning disc microscopy images of iDCs cultured in 2D suspension (top) or 3D collagen (bottom) at 48 h. p.i. GAPDH staining is shown in magenta, Vpr‐mRuby particles are shown in red, and GFP expression in productively infected iDCs is shown in green. Scale bar = 20 μm. Imaris quantification workflow. The first left panels show an example of rendered particles created with the spot finder wizard using the signal from mRuby2. The second panels show an example of segmented cells (in green, purple, and violet) obtained through the cell finder wizard using the staining of cytoplasmic GAPDH. Phalloidin‐atto 390 signal, staining F‐actin, was used to find cell surface (represented in glass transparent color) and, finally, distance of each particle with respect to the cell cytoplasm, and surface was calculated. On the top, raw data are shown, while on the bottom, the rendering is shown. The last panel shows an example of cytoplasmic, surface‐associated, and extracellular particles in blue, yellow, and red, respectively. Scale bar = 4 μm. Subcellular localization of Vpr‐mRuby2 particles in iDCs cultured in 2D suspension (left) or 3D collagen (right) for a representative donor, calculated following the workflow described in (D). For each time point, two to four pictures were analyzed and the graphs show mean values ± SD. Source data are available online for this figure.

Article Snippet: The following antibodies for the detection of cell surface markers were used at the indicated dilution: CD86 ANTIBODY, anti‐human, FITC, REAfinityTM (Miltenyi Biotec 130‐116‐262 1:100); CD86 antibody, anti‐human, PerCP‐Vio® 700, REAfinityTM (Miltenyi Biotec 130‐116‐164 1:100); CD80 antibody, anti‐human, PE (Miltenyi Biotec 130‐117‐683 1:100); PE mouse anti‐human CD83 (BD PharmigenTM 556855 1:20); PE/cyanine7 anti‐human CD1a antibody (Biolegend 300121 1:100); CD169 (Siglec‐1) antibody, anti‐human, PerCP‐Vio® 700, REAfinityTM (Miltenyi Biotec 130‐101‐508 1:100); BV421 anti‐human CD169 (Biolegend 346018 1:100); BV421 mouse anti‐human CD206 (BD Pharmigen 564062 1:20); FITC ##man CD209 (DC‐SIGN; BD PharmingenTM 551264 1:100); PerCP/cyanine5.5 anti‐human CD1c antibody (Biolegend 331514 1:50); Brilliant Violet 421TM anti‐human CD141 (Thrombomodulin) antibody (Biolegend 344113 1:50); CD327 (Siglec‐6) Antibody, anti‐human, FITC, REAfinityTM (Miltenyi Biotec 130‐112‐898 1:50); BV605 mouse anti‐human CD11c (Biolegend 301636 1:50); FITC anti‐human CD195 (CCR5) Antibody (Biolegend 359120 1:100); and PerCP/cyanine5.5 anti‐human CD4 Antibody (Biolegend 300530 1:100).

Techniques: Confocal Microscopy, Staining, Infection, Produced, Activity Assay, Virus, Microscopy, Cell Culture, Suspension, Expressing

Journal: EMBO Reports

Article Title: Tissue‐like environments shape functional interactions of HIV ‐1 with immature dendritic cells

doi: 10.15252/embr.202356818

Figure Lengend Snippet: A–E iDCs differentiated from peripheral blood monocytes for 6 days were cultured in 3D collagen or 2D suspension for 2 days and the surface expression of CD4 (A), CCR5 (B), DC‐SIGN (C), SIGLEC‐1 (D), and MMR (E) was analyzed by FACS. Representative histograms of FACS analysis showing fluorescence intensity for each receptor analyzed (Top). The graph shows the ratio between the median fluorescence intensity of the stained sample and the unstained sample for cells from three to six donors (bottom). Bars represent mean ± SD. *** P < 0.001; ** P < 0.01 calculated with paired t ‐test. F Representative images showing sum slice z‐projections. Images from the DC‐SIGN channel of cells kept in 2D suspension or 3D collagen were identically processed and are shown with identical brightness/contrast settings. The actin signal was adjusted according to its brightness in each image individually to identify the cells. Scale bar = 20 μm. G The graph shows the fluorescence intensity mean of the DC‐SIGN channel from cells grown in 2D suspension or 3D collagen. Bars define the mean ± SD and dots represent individual cells analyzed. N of cells in 2D suspension = 34; N of cells in 3D collagen = 54. *** P < 0.001 calculated with Mann–Whitney test. H–J iDCs were spin transduced with VLPs carrying Vpxmac239, 1 day prior to infection. Cells were incubated with anti‐DC‐SIGN and anti‐MMR‐blocking antibody or isotype control for 30 min, infected with HIV‐1 R5 Vpr‐Blam for 4 h to assess fusion or for 48 h to assess productive infection. (H) Representative dot plots of FACS analysis to quantify the formation of the CCF2 product generated by B‐lactamase enzymatic cleavage at 4 h.p.i. The fluorescence intensity of the CCF2 substrate is plotted against the fluorescence intensity of the CCF2 product. Gates show the percentage of CCF2 product‐positive cells. (I) Percentage of positive cells for the presence of the CFF2 product at 4 h.p.i. (J) Percentage of p24 + cells at 48 h.p.i. The graphs show mean values ± SD for cells from three donors. * P < 0.05 calculated with two‐way ANOVA test. Source data are available online for this figure.

Article Snippet: The following antibodies for the detection of cell surface markers were used at the indicated dilution: CD86 ANTIBODY, anti‐human, FITC, REAfinityTM (Miltenyi Biotec 130‐116‐262 1:100); CD86 antibody, anti‐human, PerCP‐Vio® 700, REAfinityTM (Miltenyi Biotec 130‐116‐164 1:100); CD80 antibody, anti‐human, PE (Miltenyi Biotec 130‐117‐683 1:100); PE mouse anti‐human CD83 (BD PharmigenTM 556855 1:20); PE/cyanine7 anti‐human CD1a antibody (Biolegend 300121 1:100); CD169 (Siglec‐1) antibody, anti‐human, PerCP‐Vio® 700, REAfinityTM (Miltenyi Biotec 130‐101‐508 1:100); BV421 anti‐human CD169 (Biolegend 346018 1:100); BV421 mouse anti‐human CD206 (BD Pharmigen 564062 1:20); FITC ##man CD209 (DC‐SIGN; BD PharmingenTM 551264 1:100); PerCP/cyanine5.5 anti‐human CD1c antibody (Biolegend 331514 1:50); Brilliant Violet 421TM anti‐human CD141 (Thrombomodulin) antibody (Biolegend 344113 1:50); CD327 (Siglec‐6) Antibody, anti‐human, FITC, REAfinityTM (Miltenyi Biotec 130‐112‐898 1:50); BV605 mouse anti‐human CD11c (Biolegend 301636 1:50); FITC anti‐human CD195 (CCR5) Antibody (Biolegend 359120 1:100); and PerCP/cyanine5.5 anti‐human CD4 Antibody (Biolegend 300530 1:100).

Techniques: Cell Culture, Suspension, Expressing, Fluorescence, Staining, MANN-WHITNEY, Transduction, Infection, Incubation, Blocking Assay, Control, Generated

Journal: Frontiers in Oncology

Article Title: Rational Combination of Parvovirus H1 With CTLA-4 and PD-1 Checkpoint Inhibitors Dampens the Tumor Induced Immune Silencing

doi: 10.3389/fonc.2019.00425

Figure Lengend Snippet: Determination of DC maturation after co-culturing of iDCs with uninfected and H-1PV infected Sk29Mel-1 cells with or without the checkpoint inhibitors ipilimumab and nivolumab for 3 days. Surface expression of the maturation markers CD80, CD83, and CD86 was determined using FACS analysis (A) while release of IL-6, IFNγ, and TNFα in the supernatant is determined by ELISA (B–D) . Experiments were performed independently as triplicate * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. iDC, immatured dendritic cells; mDC, matured dendritic cells; Ipi, ipilimumab; Nivo, nivolumab; ELISA, enzyme linked immunosorbent assay; FACS, fluorescence-activated cell sorting.

Article Snippet: After 3 days, the maturation status was determined by extracellular staining and measurement of CD80, CD83, and CD86 (Becton Dickinson; Heidelberg, Germany) via fluorescence-activated cell sorting (FACS) and by measurement of the interleukin-6 (IL-6), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) levels in the supernatant via an enzyme linked immunosorbent assay (ELISA; Thermo Fisher Scientific, Darmstadt, Germany).

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, FACS

Journal: Frontiers in Oncology

Article Title: Rational Combination of Parvovirus H1 With CTLA-4 and PD-1 Checkpoint Inhibitors Dampens the Tumor Induced Immune Silencing

doi: 10.3389/fonc.2019.00425

Figure Lengend Snippet: Determination of DC maturation after co-culturing of iDCs with uninfected and H-1PV infected Mz7Mel with or without the checkpoint inhibitors ipilimumab and nivolumab for 3 days. Results of three different experiments are shown. Surface expression of the maturation marker CD80, CD83 and CD86 was determined using FACS analysis (A) while release of IL-6, IFNγ, and TNFα in the supernatant is determined by ELISA (B–D) . Experiments were performed independently as triplicate * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. iDC, immatured dendritic cells; mDC, matured dendritic cells; Ipi, ipilimumab; Nivo, nivolumab; ELISA, enzyme linked immunosorbent assay; FACS, fluorescence-activated cell sorting.

Article Snippet: After 3 days, the maturation status was determined by extracellular staining and measurement of CD80, CD83, and CD86 (Becton Dickinson; Heidelberg, Germany) via fluorescence-activated cell sorting (FACS) and by measurement of the interleukin-6 (IL-6), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) levels in the supernatant via an enzyme linked immunosorbent assay (ELISA; Thermo Fisher Scientific, Darmstadt, Germany).

Techniques: Infection, Expressing, Marker, Enzyme-linked Immunosorbent Assay, Fluorescence, FACS