cd209 pe Search Results


anti dc sign pe  (R&D Systems)
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Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living <t>cells,</t> <t>CD3</t> + T cells (top and middle panel), or <t>DC-SIGN</t> + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.
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Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living <t>cells,</t> <t>CD3</t> + T cells (top and middle panel), or <t>DC-SIGN</t> + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.
Mouse Anti Human Dc Sign Dcn47 5, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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monoclonal anti human dc sign phycoerythrin  (R&D Systems)
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Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living <t>cells,</t> <t>CD3</t> + T cells (top and middle panel), or <t>DC-SIGN</t> + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.
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Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living <t>cells,</t> <t>CD3</t> + T cells (top and middle panel), or <t>DC-SIGN</t> + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.
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cd209 pe  (Miltenyi Biotec)
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Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living <t>cells,</t> <t>CD3</t> + T cells (top and middle panel), or <t>DC-SIGN</t> + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.
Cd209 Pe, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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monoclonal antibody 120507  (R&D Systems)
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Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living <t>cells,</t> <t>CD3</t> + T cells (top and middle panel), or <t>DC-SIGN</t> + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.
Monoclonal Antibody 120507, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse igg2b pe-conjugated antibody  (Bio-Techne corporation)
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Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living <t>cells,</t> <t>CD3</t> + T cells (top and middle panel), or <t>DC-SIGN</t> + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.
Mouse Igg2b Pe Conjugated Antibody, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pe conjugated anti cd209 dc sign  (R&D Systems)
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Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living <t>cells,</t> <t>CD3</t> + T cells (top and middle panel), or <t>DC-SIGN</t> + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.
Pe Conjugated Anti Cd209 Dc Sign, supplied by R&D Systems, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living <t>cells,</t> <t>CD3</t> + T cells (top and middle panel), or <t>DC-SIGN</t> + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.
Intercellular Adhesion Molecule 3 Grabbing Non Integrin Dc Sign Pe Fab8345p, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living cells, CD3 + T cells (top and middle panel), or DC-SIGN + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Dendritic Cell Maturation Regulates TSPAN7 Function in HIV-1 Transfer to CD4 + T Lymphocytes

doi: 10.3389/fcimb.2020.00070

Figure Lengend Snippet: Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living cells, CD3 + T cells (top and middle panel), or DC-SIGN + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.

Article Snippet: For flow cytometry analysis, the staining was performed with anti-CD3 AF700 (for T cells) (eBioscience; 56-0038-82), anti–DC-SIGN PE (for MDDCs) (Clone, 120507; R&D systems, catalog number: FAB161P), anti-CD169 APC (for CD169/Siglec-1; Biolegend; 346007), and DAPI for live/dead gating and in some cases anti-P24 PE (Beckman Coulter, 6604667).

Techniques: Expressing, Flow Cytometry, Infection, Co-Culture Assay, Real-time Polymerase Chain Reaction

CD169, as an HIV-1 receptor, mostly impacts transfer from mature MDDCs rather than immature MDDCs. (A) Flow cytometry plots showing CD86, DC-SIGN, and CD169 expression levels on MDDCs (pre-gated on SSC FSC, living cells, CD3 − cells and singlets). Panels show the expression of these proteins in iMDDCs (left panel) and MDDCs with LPS pretreatment at 100 ng/ml for 48 or 24 h before co-culture (middle and right panels, respectively). (B) Percentage of variation of HIV-1 transfer when using iMDDCs or LPS-treated MDDCs (100 ng/ml LPS for different lengths of time) incubated with a blocking antibody against CD169 as compared to an isotype control for each condition. Results are displayed for 4 different blood donors with the mean ± SD of technical triplicates. (C) Percent of variation in HIV-1 transfer to assess the impact of blocking CD169 and TSPAN7 knockdown as compared to scramble shRNA on MDDCs matured with LPS for 48 h treated by an isotype control. Mean ± SD of seven different blood donors in 4 experiments. (B,C) NS, not significant. ** p < 0.01; *** p < 0.001. (D) Confocal microscopy images of iMDDCs (left panel) and mature MDDCs (mMDDCs) right panel, to assess the degree of colocalization between CD169 (magenta) and incoming X4–HIV-1–Gag-iGFP (green). Actin filaments and nuclei were stained with phalloidin (red) and DAPI (blue). Four hundred nanometers of Z-stacks were taken 40 h after the start of the co-culture with CD4 + T cells and X4–HIV-1–Gag-iGFP. The pictures presented here are from a representative donor from four unrelated blood donors.

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Dendritic Cell Maturation Regulates TSPAN7 Function in HIV-1 Transfer to CD4 + T Lymphocytes

doi: 10.3389/fcimb.2020.00070

Figure Lengend Snippet: CD169, as an HIV-1 receptor, mostly impacts transfer from mature MDDCs rather than immature MDDCs. (A) Flow cytometry plots showing CD86, DC-SIGN, and CD169 expression levels on MDDCs (pre-gated on SSC FSC, living cells, CD3 − cells and singlets). Panels show the expression of these proteins in iMDDCs (left panel) and MDDCs with LPS pretreatment at 100 ng/ml for 48 or 24 h before co-culture (middle and right panels, respectively). (B) Percentage of variation of HIV-1 transfer when using iMDDCs or LPS-treated MDDCs (100 ng/ml LPS for different lengths of time) incubated with a blocking antibody against CD169 as compared to an isotype control for each condition. Results are displayed for 4 different blood donors with the mean ± SD of technical triplicates. (C) Percent of variation in HIV-1 transfer to assess the impact of blocking CD169 and TSPAN7 knockdown as compared to scramble shRNA on MDDCs matured with LPS for 48 h treated by an isotype control. Mean ± SD of seven different blood donors in 4 experiments. (B,C) NS, not significant. ** p < 0.01; *** p < 0.001. (D) Confocal microscopy images of iMDDCs (left panel) and mature MDDCs (mMDDCs) right panel, to assess the degree of colocalization between CD169 (magenta) and incoming X4–HIV-1–Gag-iGFP (green). Actin filaments and nuclei were stained with phalloidin (red) and DAPI (blue). Four hundred nanometers of Z-stacks were taken 40 h after the start of the co-culture with CD4 + T cells and X4–HIV-1–Gag-iGFP. The pictures presented here are from a representative donor from four unrelated blood donors.

Article Snippet: For flow cytometry analysis, the staining was performed with anti-CD3 AF700 (for T cells) (eBioscience; 56-0038-82), anti–DC-SIGN PE (for MDDCs) (Clone, 120507; R&D systems, catalog number: FAB161P), anti-CD169 APC (for CD169/Siglec-1; Biolegend; 346007), and DAPI for live/dead gating and in some cases anti-P24 PE (Beckman Coulter, 6604667).

Techniques: Flow Cytometry, Expressing, Co-Culture Assay, Incubation, Blocking Assay, shRNA, Confocal Microscopy, Staining