surface markers cd86  (ATCC)

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: Journal of Orthopaedic Translation

Article Title: An aptamer specifically targeting mCRP ameliorates experimental arthritis

doi: 10.1016/j.jot.2025.08.010

Figure Lengend Snippet: Development of CAIA in hepatocyte-specific CRP knockout mice. (A) Schematic diagram illustrating the development of CAIA in CRP +/+ and CRP ΔHep/ΔHep mice. (B) Arthritis scoring of CRP +/+ and CRP ΔHep/ΔHep mice with or without CAIA induction. Mice without CAIA served as non-immunized (NI) controls. (C and D) Serum IL-6 (C) and IL-1β (D) levels in CRP +/+ and CRP ΔHep/ΔHep mice with or without CAIA induction, as determined by ELISA. (E) Photographs of hind paws of CRP +/+ and CRP ΔHep/ΔHep mice with or without CAIA induction. (F) H&E staining of paw sections of CRP +/+ and CRP ΔHep/ΔHep mice with or without CAIA induction. Scale bar = 300 μm. (G) SO&FG staining of paw sections of CRP +/+ and CRP ΔHep/ΔHep mice with or without CAIA induction. Scale bar = 300 μm. (H) IF staining of paw sections of CRP +/+ and CRP ΔHep/ΔHep mice with or without CAIA induction, using an anti-Vimentin, an anti-CD86, or an anti-MMP-13 antibody. Scale bar = 50 μm. (I – K) Quantification of expression of Vimentin (I) , CD86 (J), and MMP-13 (K) on IF-stained sections. Data are represented as mean ± SD of n = 5 per group. P-values from one-way ANOVA (C, D, I-K) or two-way ANOVA (B): ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Surface marker CD86 was stained directly with anti-human CD86 antibody (Proteintech, Cat# 13395-1-AP, CN).

Techniques: Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: An aptamer specifically targeting mCRP ameliorates experimental arthritis

doi: 10.1016/j.jot.2025.08.010

Figure Lengend Snippet: Establishment of surgical DMM in hepatocyte-specific CRP knockout mice. (A) Schematic diagram illustrating the establishment of surgical DMM in CRP +/+ and CRP ΔHep/ΔHep mice. (B) μCT scans of knee joints of CRP +/+ and CRP ΔHep/ΔHep mice after sham or DMM surgery, with black arrows indicating the osteophyte formation. Scale bar = 1 mm. (C) H&E staining of joint sections of CRP +/+ and CRP ΔHep/ΔHep mice after sham or DMM surgery. Scale bar = 300 μm. (D) SO&FG staining of joint sections of CRP +/+ and CRP ΔHep/ΔHep mice after sham or DMM surgery. Scale bar = 300 μm. (E) IF staining of joint sections of CRP +/+ and CRP ΔHep/ΔHep mice after sham or DMM surgery, using an anti-vimentin, an anti-CD86, or an anti-COL2A1 antibody. Scale bar = 50 μm. (F – I) Quantification of OARSI score (F) , cartilage area (G) , osteophyte score (H), and synovitis score (I) of CRP +/+ and CRP ΔHep/ΔHep mice after sham or DMM surgery. (J – L) Quantification of expression of vimentin (J) , CD86 (K), and COL2A1 (L) on IF-stained sections. Data are represented as mean ± SD of n = 5 per group. P-values from one-way ANOVA (F–L): ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Surface marker CD86 was stained directly with anti-human CD86 antibody (Proteintech, Cat# 13395-1-AP, CN).

Techniques: Knock-Out, Staining, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: An aptamer specifically targeting mCRP ameliorates experimental arthritis

doi: 10.1016/j.jot.2025.08.010

Figure Lengend Snippet: Therapeutic effects of ApmCRP3 in CIA mice. (A) Schematic diagram of experimental design using CIA mice. Briefly, CIA mice were treated with PBS, 1,6-bisPC, NC, or ApmCRP3 every 3 days for 45 days. NC or ApmCRP3 was intravenously injected at a dose of 25 mg/kg, and 1,6-bisPC was intraperitoneally administered at a dose of 3 mg/kg. (B) Arthritis scoring of NI or CIA mice from different treatment groups. (C) μCT scans of hind paws of NI or CIA mice from different treatment groups, with black arrows indicating the bone erosion. (D) H&E staining of paw sections of NI or CIA mice from different treatment groups. Scale bar = 300 μm. (E) SO&FG staining of paw sections of NI or CIA mice from different treatment groups. Scale bar = 300 μm. (F) IF staining of paw sections of NI or CIA mice from different treatment groups, using an anti-vimentin, an anti-CD86, or an anti-MMP-13 antibody. Scale bar = 50 μm. (G – I) Quantification of expression of Vimentin (G) , CD86 (H) , and MMP-13 (I) on IF-stained sections. Data were represented as mean ± SD of n = 6 per group. P-values from one-way ANOVA (G–I) or two-way ANOVA (B): ∗p < 0.05, ∗∗∗p < 0.001.

Article Snippet: Surface marker CD86 was stained directly with anti-human CD86 antibody (Proteintech, Cat# 13395-1-AP, CN).

Techniques: Injection, Staining, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: An aptamer specifically targeting mCRP ameliorates experimental arthritis

doi: 10.1016/j.jot.2025.08.010

Figure Lengend Snippet: Therapeutic effects of ApmCRP3 in DMM mice. (A) Schematic diagram of experimental design using DMM mice. Briefly, DMM mice were treated with PBS, 1,6-bisPC, NC, or ApmCRP3 every 3 days for 49 days. NC or ApmCRP3 was intravenously injected at a dose of 25 mg/kg, and 1,6-bisPC was intraperitoneally administered at a dose of 3 mg/kg. (B) μCT scans of knee joints of sham or DMM mice from different treatment groups, with black arrows indicating the osteophyte formation. (C) H&E staining of joint sections of sham or DMM mice from different treatment groups. Scale bar = 300 μm. (D) SO&FG staining of joint sections of sham or DMM mice from different treatment groups. Scale bar = 300 μm. (E) IF staining of joint sections of sham or DMM mice from different treatment groups, using an anti-vimentin, an anti-CD86, or an anti-COL2A1 antibody. Scale bar = 50 μm. (F – I) Quantification of OARSI score (F) , cartilage area (G) , osteophyte score (H), and synovitis score (I) of sham or DMM mice from different treatment groups. (J – L) Quantification of expression of vimentin (J) , CD86 (K), and COL2A1 (L) on IF-stained sections. Data were represented as mean ± SD of n = 6 per group. P-values from one-way ANOVA (F–L): ∗∗∗p < 0.001.

Article Snippet: Surface marker CD86 was stained directly with anti-human CD86 antibody (Proteintech, Cat# 13395-1-AP, CN).

Techniques: Injection, Staining, Expressing