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HiMedia Laboratories hiv 1 transfer assay
HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of <t>HIV-1</t> (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P
Hiv 1 Transfer Assay, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission"

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2016.00600

HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P
Figure Legend Snippet: HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P

Techniques Used: Expressing, Incubation, Binding Assay, Cell Culture

Diagrammatic model explaining the possible implications of the tripartite molecular interplay between DC-SIGN, C1q, and gC1qR . (A) By virtue of its ability to bind to DC-SIGN on the cell surface, C1q is likely to inhibit interaction between DC-SIGN and HIV-1 gp120, resulting in the inhibition of viral transfer. (B) On the DC/Monocyte surface, a trimolecular receptor complex is formed between gC1qR, C1q, and DC-SIGN. Although each of these molecules can bind the HIV-1 virus independently, we postulate that it is the binding of the HIV-1 gp-41 to both gC1qR and C1q that initiates the membrane fusion before the final binding of gp120 to DC-SIGN and/or CD4, eventually allowing the internalization of the virus. It is possible that HIV-1 interaction with DC/monocytes causes recruitment of gC1qR to the cell surface, or its secretion, which in turn, can bind to C1q globular heads, thereby neutralizing the protection offered by C1q.
Figure Legend Snippet: Diagrammatic model explaining the possible implications of the tripartite molecular interplay between DC-SIGN, C1q, and gC1qR . (A) By virtue of its ability to bind to DC-SIGN on the cell surface, C1q is likely to inhibit interaction between DC-SIGN and HIV-1 gp120, resulting in the inhibition of viral transfer. (B) On the DC/Monocyte surface, a trimolecular receptor complex is formed between gC1qR, C1q, and DC-SIGN. Although each of these molecules can bind the HIV-1 virus independently, we postulate that it is the binding of the HIV-1 gp-41 to both gC1qR and C1q that initiates the membrane fusion before the final binding of gp120 to DC-SIGN and/or CD4, eventually allowing the internalization of the virus. It is possible that HIV-1 interaction with DC/monocytes causes recruitment of gC1qR to the cell surface, or its secretion, which in turn, can bind to C1q globular heads, thereby neutralizing the protection offered by C1q.

Techniques Used: Inhibition, Binding Assay

Competitive inhibition of DC-SIGN: HIV-1 gp120 interaction by globular head modules and gC1qR . (A) ELISA to assess whether gC1qR and ghB directly compete for the same binding site on DC-SIGN: DC-SIGN was coated at 5 µg/well overnight at 4°C. Wells were blocked with 2% BSA in PBS for 2 h at 37°C. gC1qR (5 µg/well) and different concentrations of ghB (5, 2.5, 1.25, 0.625 µg/well) were added in buffer containing 5 mM CaCl 2 . Incubation was carried out at 37°C for 1.5 h and 4°C for 1.5 h. Following repeated washes, bound gC1qR was probed using rabbit anti-gC1qR polyclonal antibodies (1:1,000) and Protein A-HRP (1:1,000). Color was developed using o -phenylenediamine dihydrochloride substrate; (B) competition between DC-SIGN tetramer and C1q globular head modules to bind solid-phase gp120. Microtiter wells were coated with 250 ng of gp120. Various concentrations of ghA, ghB, ghC, and C1q and constant 2.5 µg/mL of DC-SIGN were incubated at 37°C for 1 h and then at 4°C for 1 h. The binding of DC-SIGN to gp120 in the presence of globular heads or C1q was detected using rabbit anti-DC antibody (1:500), probed with Protein A HRP (1:5,000). DC-SIGN alone binding to gp120 was used as 100%.
Figure Legend Snippet: Competitive inhibition of DC-SIGN: HIV-1 gp120 interaction by globular head modules and gC1qR . (A) ELISA to assess whether gC1qR and ghB directly compete for the same binding site on DC-SIGN: DC-SIGN was coated at 5 µg/well overnight at 4°C. Wells were blocked with 2% BSA in PBS for 2 h at 37°C. gC1qR (5 µg/well) and different concentrations of ghB (5, 2.5, 1.25, 0.625 µg/well) were added in buffer containing 5 mM CaCl 2 . Incubation was carried out at 37°C for 1.5 h and 4°C for 1.5 h. Following repeated washes, bound gC1qR was probed using rabbit anti-gC1qR polyclonal antibodies (1:1,000) and Protein A-HRP (1:1,000). Color was developed using o -phenylenediamine dihydrochloride substrate; (B) competition between DC-SIGN tetramer and C1q globular head modules to bind solid-phase gp120. Microtiter wells were coated with 250 ng of gp120. Various concentrations of ghA, ghB, ghC, and C1q and constant 2.5 µg/mL of DC-SIGN were incubated at 37°C for 1 h and then at 4°C for 1 h. The binding of DC-SIGN to gp120 in the presence of globular heads or C1q was detected using rabbit anti-DC antibody (1:500), probed with Protein A HRP (1:5,000). DC-SIGN alone binding to gp120 was used as 100%.

Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation

2) Product Images from "Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission"

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2016.00600

HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P
Figure Legend Snippet: HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P

Techniques Used: Expressing, Incubation, Binding Assay, Cell Culture

3) Product Images from "Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission"

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2016.00600

HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P
Figure Legend Snippet: HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P

Techniques Used: Expressing, Incubation, Binding Assay, Cell Culture

Related Articles

Recombinant:

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission
Article Snippet: .. HIV-1 Transfer Assay with DC-HEK Cells and Pooled Peripheral Blood Mononuclear Cell (PBMC) Pooled peripheral blood mononuclear cells (HiMedia Laboratories, India) were cultured in RPMI 1640 medium (Sigma Aldrich) containing 10% FBS, 1% Penicillin–Streptomycin (Complete RPMI medium), and stimulated with 5 µg/mL phytohemaglutinin (PHA) and 10 U/Ml of recombinant-human IL-2 (Gibco) for 24 h. PHA/IL-2 was washed off and activated PBMCs were cultured further for 3 days in complete RPMI 1640 medium. .. DC-HEK cells were grown and maintained in DMEM-F12 (Sigma-Aldrich, USA) containing 10% FBS and blasticidin (5 µg/mL) (Gibco).

Cell Culture:

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission
Article Snippet: .. HIV-1 Transfer Assay with DC-HEK Cells and Pooled Peripheral Blood Mononuclear Cell (PBMC) Pooled peripheral blood mononuclear cells (HiMedia Laboratories, India) were cultured in RPMI 1640 medium (Sigma Aldrich) containing 10% FBS, 1% Penicillin–Streptomycin (Complete RPMI medium), and stimulated with 5 µg/mL phytohemaglutinin (PHA) and 10 U/Ml of recombinant-human IL-2 (Gibco) for 24 h. PHA/IL-2 was washed off and activated PBMCs were cultured further for 3 days in complete RPMI 1640 medium. .. DC-HEK cells were grown and maintained in DMEM-F12 (Sigma-Aldrich, USA) containing 10% FBS and blasticidin (5 µg/mL) (Gibco).

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    HiMedia Laboratories pooled peripheral blood mononuclear cell pbmc pooled peripheral blood mononuclear cells
    <t>HIV</t> transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated <t>PBMCs</t> for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P
    Pooled Peripheral Blood Mononuclear Cell Pbmc Pooled Peripheral Blood Mononuclear Cells, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pooled peripheral blood mononuclear cell pbmc pooled peripheral blood mononuclear cells/product/HiMedia Laboratories
    Average 90 stars, based on 1 article reviews
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    HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P

    Journal: Frontiers in Immunology

    Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

    doi: 10.3389/fimmu.2016.00600

    Figure Lengend Snippet: HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P

    Article Snippet: HIV-1 Transfer Assay with DC-HEK Cells and Pooled Peripheral Blood Mononuclear Cell (PBMC) Pooled peripheral blood mononuclear cells (HiMedia Laboratories, India) were cultured in RPMI 1640 medium (Sigma Aldrich) containing 10% FBS, 1% Penicillin–Streptomycin (Complete RPMI medium), and stimulated with 5 µg/mL phytohemaglutinin (PHA) and 10 U/Ml of recombinant-human IL-2 (Gibco) for 24 h. PHA/IL-2 was washed off and activated PBMCs were cultured further for 3 days in complete RPMI 1640 medium.

    Techniques: Expressing, Incubation, Binding Assay, Cell Culture