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lipopolysaccharide binding protein lbp  (R&D Systems)


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    R&D Systems lipopolysaccharide binding protein lbp
    A HUVECs were stimulated with ECVE nic or control (EBM-2) for 12 hours (incubation). Then, 100 ng/ml LPS with co-factors <t>LBP</t> and CD14 or co-factors alone (sham) were added and incubated for another 18 hours. Created in BioRender. Stahl, K. (2026) https://BioRender.com/nu4iwxz . B Angpt-2 concentrations in the supernatant were measured by ELISA when LPS was added as well as 12 and 18 hours after LPS or sham stimulation and are shown over time. Each group consisted of n =6. Mixed ANOVA with according post-tests were performed. C 18 hours after stimulation with LPS or sham Angpt-2 mRNA transcription was measured by qPCR. Each group consisted of n =9. D Additionally, the Angpt-2/Tie2 mRNA ratio is shown after initial ECVE nic incubation and 18 hours of LPS or sham stimulation. ECVE nic / sham consisted of n=8, all other groups consisted of n =9. Groups were analyzed by Kruskal-Wallis test with post-hoc Dunn test. E TER was measured at different time points by an ECIS system. HUVECs were pre-incubated with ECVE nic ( n =7) or control ( n =8) for 12 hours. Then half of the medium was changed and cells were stimulated with LPS. Results are shown as dot plots with mean ± SD. A linear mixed-effects model was used for analysis. P -values < 0.05 are considered significant. Abbreviations: Angpt-2 - Angiopoietin-2, ECIS - Electric cell-substrate impedance sensing, ECVE – e-cigarette vapor extract, HUVECs – human umbilical vein endothelial cells, LPS - <t>lipopolysaccharide,</t> SD – standard deviation, TER – transendothelial electrical resistance
    Lipopolysaccharide Binding Protein Lbp, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/recombinant+lbp/Recombinant+Human+LBP+Protein/pmc13621675-44-26-31
    Average 92 stars, based on 37 article reviews
    lipopolysaccharide binding protein lbp - by Bioz Stars, 2026-10
    92/100 stars

    Images

    1) Product Images from "The Tie-2 agonist PMC-403 inhibits endothelial damage induced by e-cigarette vapor or LPS"

    Article Title: The Tie-2 agonist PMC-403 inhibits endothelial damage induced by e-cigarette vapor or LPS

    Journal: Respiratory Research

    doi: 10.1186/s12931-026-03928-4

    A HUVECs were stimulated with ECVE nic or control (EBM-2) for 12 hours (incubation). Then, 100 ng/ml LPS with co-factors LBP and CD14 or co-factors alone (sham) were added and incubated for another 18 hours. Created in BioRender. Stahl, K. (2026) https://BioRender.com/nu4iwxz . B Angpt-2 concentrations in the supernatant were measured by ELISA when LPS was added as well as 12 and 18 hours after LPS or sham stimulation and are shown over time. Each group consisted of n =6. Mixed ANOVA with according post-tests were performed. C 18 hours after stimulation with LPS or sham Angpt-2 mRNA transcription was measured by qPCR. Each group consisted of n =9. D Additionally, the Angpt-2/Tie2 mRNA ratio is shown after initial ECVE nic incubation and 18 hours of LPS or sham stimulation. ECVE nic / sham consisted of n=8, all other groups consisted of n =9. Groups were analyzed by Kruskal-Wallis test with post-hoc Dunn test. E TER was measured at different time points by an ECIS system. HUVECs were pre-incubated with ECVE nic ( n =7) or control ( n =8) for 12 hours. Then half of the medium was changed and cells were stimulated with LPS. Results are shown as dot plots with mean ± SD. A linear mixed-effects model was used for analysis. P -values < 0.05 are considered significant. Abbreviations: Angpt-2 - Angiopoietin-2, ECIS - Electric cell-substrate impedance sensing, ECVE – e-cigarette vapor extract, HUVECs – human umbilical vein endothelial cells, LPS - lipopolysaccharide, SD – standard deviation, TER – transendothelial electrical resistance
    Figure Legend Snippet: A HUVECs were stimulated with ECVE nic or control (EBM-2) for 12 hours (incubation). Then, 100 ng/ml LPS with co-factors LBP and CD14 or co-factors alone (sham) were added and incubated for another 18 hours. Created in BioRender. Stahl, K. (2026) https://BioRender.com/nu4iwxz . B Angpt-2 concentrations in the supernatant were measured by ELISA when LPS was added as well as 12 and 18 hours after LPS or sham stimulation and are shown over time. Each group consisted of n =6. Mixed ANOVA with according post-tests were performed. C 18 hours after stimulation with LPS or sham Angpt-2 mRNA transcription was measured by qPCR. Each group consisted of n =9. D Additionally, the Angpt-2/Tie2 mRNA ratio is shown after initial ECVE nic incubation and 18 hours of LPS or sham stimulation. ECVE nic / sham consisted of n=8, all other groups consisted of n =9. Groups were analyzed by Kruskal-Wallis test with post-hoc Dunn test. E TER was measured at different time points by an ECIS system. HUVECs were pre-incubated with ECVE nic ( n =7) or control ( n =8) for 12 hours. Then half of the medium was changed and cells were stimulated with LPS. Results are shown as dot plots with mean ± SD. A linear mixed-effects model was used for analysis. P -values < 0.05 are considered significant. Abbreviations: Angpt-2 - Angiopoietin-2, ECIS - Electric cell-substrate impedance sensing, ECVE – e-cigarette vapor extract, HUVECs – human umbilical vein endothelial cells, LPS - lipopolysaccharide, SD – standard deviation, TER – transendothelial electrical resistance

    Techniques Used: Control, Incubation, Enzyme-linked Immunosorbent Assay, Analysis, Electric Cell-substrate Impedance Sensing, Standard Deviation

    Related Articles

    Sterility:

    Article Title: Lipopolysaccharide stimulates platelets through an IL-1β autocrine loop
    Article Snippet: .. Chemicals and reagents were purchased from the following sources: sterile filtered HBSS and M199 (BioWhittaker); sterile tissue culture plates, (Falcon Labware); human serum albumin, (Baxter Healthcare); endotoxin-free PBS, phenol-extracted LPS ( Escherichia coli O111:B4) that is free of lipoprotein contamination (List Biological Laboratories); recombinant soluble CD14, recombinant LBP, and IL-1β ELISA kits, (R&D Systems); Caspase-1 inhibitors z-WEHD-FMK (FMK002) and z-YVAD-FMK (FMK005) were obtained from R&D Systems. .. Alexa 594 -conjugated wheat germ agglutinin (Invitrogen), Alexa Flour 488 Goat anti rabbit (Invitrogen {"type":"entrez-nucleotide","attrs":{"text":"A11034","term_id":"489250"}} A11034 ), Rabbit polyclonal anti-IL1 Receptor 1 (Abcam ab59995), isotype APC Mouse IgG 1 (BD pharmingen 555751), rabbit anti-mouse IL-1β (Abcam Ab9722), anti-CD14 (e-Bioscience 16-0149-82), anti-β-actin (Santa Cruz Biotechnology), Goat polyclonal anti-P-selectin (C-20; SC-6941), recombinant human Interleukin 1 Receptor Antagonist (rhIL1Ra) (R&D Systems).

    Article Title: Lipopolysaccharide stimulates platelets through an IL-1β autocrine loop
    Article Snippet: .. Chemicals and reagents Chemicals and reagents were purchased from the following sources: sterile filtered HBSS and M199 (BioWhittaker); sterile tissue culture plates, (Falcon Labware); human serum albumin, (Baxter Healthcare); endotoxin-free PBS, phenol-extracted LPS ( Escherichia coli O111:B4) that is free of lipoprotein contamination (List Biological Laboratories); recombinant soluble CD14, recombinant LBP, and IL-1β ELISA kits, (R&D Systems); Caspase-1 inhibitors z-WEHD-FMK (FMK002) and z-YVAD-FMK (FMK005) were obtained from R&D Systems. .. Alexa 594 -conjugated wheat germ agglutinin (Invitrogen), Alexa Flour 488 Goat anti rabbit (Invitrogen {"type":"entrez-nucleotide","attrs":{"text":"A11034","term_id":"489250"}} A11034 ), Rabbit polyclonal anti-IL1 Receptor 1 (Abcam ab59995), isotype APC Mouse IgG 1 (BD pharmingen 555751), rabbit anti-mouse IL-1β (Abcam Ab9722), anti-CD14 (e-Bioscience 16-0149-82), anti-β-actin (Santa Cruz Biotechnology), Goat polyclonal anti-P-selectin (C-20; SC-6941), recombinant human Interleukin 1 Receptor Antagonist (rhIL1Ra) (R&D Systems).

    Recombinant:

    Article Title: Lipopolysaccharide stimulates platelets through an IL-1β autocrine loop
    Article Snippet: .. Chemicals and reagents were purchased from the following sources: sterile filtered HBSS and M199 (BioWhittaker); sterile tissue culture plates, (Falcon Labware); human serum albumin, (Baxter Healthcare); endotoxin-free PBS, phenol-extracted LPS ( Escherichia coli O111:B4) that is free of lipoprotein contamination (List Biological Laboratories); recombinant soluble CD14, recombinant LBP, and IL-1β ELISA kits, (R&D Systems); Caspase-1 inhibitors z-WEHD-FMK (FMK002) and z-YVAD-FMK (FMK005) were obtained from R&D Systems. .. Alexa 594 -conjugated wheat germ agglutinin (Invitrogen), Alexa Flour 488 Goat anti rabbit (Invitrogen {"type":"entrez-nucleotide","attrs":{"text":"A11034","term_id":"489250"}} A11034 ), Rabbit polyclonal anti-IL1 Receptor 1 (Abcam ab59995), isotype APC Mouse IgG 1 (BD pharmingen 555751), rabbit anti-mouse IL-1β (Abcam Ab9722), anti-CD14 (e-Bioscience 16-0149-82), anti-β-actin (Santa Cruz Biotechnology), Goat polyclonal anti-P-selectin (C-20; SC-6941), recombinant human Interleukin 1 Receptor Antagonist (rhIL1Ra) (R&D Systems).

    Article Title: Intestinal epithelium-derived high density lipoprotein restrains liver injury via the portal vein
    Article Snippet: The layer containing liver macrophages was plated in RPMI-1640 (Hyclone) containing 10% fetal bovine serum (FBS). .. After 2 h culture to allow for cell attachment, the cell medium was changed to “vehicle” medium, which was serum-free RPMI-1640 containing 1 μg/ml recombinant LBP (R&D systems; #6635-LP) for 3 h of culture. .. HDL preparations as described were added, or not, to these cultures with LPS (Sigma-Aldrich; #L2630) presence.

    Article Title: Lipopolysaccharide stimulates platelets through an IL-1β autocrine loop
    Article Snippet: .. Chemicals and reagents Chemicals and reagents were purchased from the following sources: sterile filtered HBSS and M199 (BioWhittaker); sterile tissue culture plates, (Falcon Labware); human serum albumin, (Baxter Healthcare); endotoxin-free PBS, phenol-extracted LPS ( Escherichia coli O111:B4) that is free of lipoprotein contamination (List Biological Laboratories); recombinant soluble CD14, recombinant LBP, and IL-1β ELISA kits, (R&D Systems); Caspase-1 inhibitors z-WEHD-FMK (FMK002) and z-YVAD-FMK (FMK005) were obtained from R&D Systems. .. Alexa 594 -conjugated wheat germ agglutinin (Invitrogen), Alexa Flour 488 Goat anti rabbit (Invitrogen {"type":"entrez-nucleotide","attrs":{"text":"A11034","term_id":"489250"}} A11034 ), Rabbit polyclonal anti-IL1 Receptor 1 (Abcam ab59995), isotype APC Mouse IgG 1 (BD pharmingen 555751), rabbit anti-mouse IL-1β (Abcam Ab9722), anti-CD14 (e-Bioscience 16-0149-82), anti-β-actin (Santa Cruz Biotechnology), Goat polyclonal anti-P-selectin (C-20; SC-6941), recombinant human Interleukin 1 Receptor Antagonist (rhIL1Ra) (R&D Systems).

    Article Title: Intestinal epithelium-derived high density lipoprotein restrains liver injury via the portal vein
    Article Snippet: For sandwich ELISA to analyze LPS-HDL binding, we used high-binding clear polystyrenes microplates (R&D systems, DY990), and purified HDL was immobilized for 2 h on these plates at 37°C at 10 μg/ml. .. After washing, plates were blocked with 1% BSA for 1 h. Then Biotin-LPS (InvivoGen; #tlrl-lpsbiot) was pre-incubated with or without 1 μg/ml recombinant LBP (R&D Systems) for 1 h and incubated in plates for 30 min. Streptavidin peroxidase (R&D Systems) was added, followed by diaminobenzidine substrate (Abcam), for colorimetric reactions. ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: Lipopolysaccharide stimulates platelets through an IL-1β autocrine loop
    Article Snippet: .. Chemicals and reagents were purchased from the following sources: sterile filtered HBSS and M199 (BioWhittaker); sterile tissue culture plates, (Falcon Labware); human serum albumin, (Baxter Healthcare); endotoxin-free PBS, phenol-extracted LPS ( Escherichia coli O111:B4) that is free of lipoprotein contamination (List Biological Laboratories); recombinant soluble CD14, recombinant LBP, and IL-1β ELISA kits, (R&D Systems); Caspase-1 inhibitors z-WEHD-FMK (FMK002) and z-YVAD-FMK (FMK005) were obtained from R&D Systems. .. Alexa 594 -conjugated wheat germ agglutinin (Invitrogen), Alexa Flour 488 Goat anti rabbit (Invitrogen {"type":"entrez-nucleotide","attrs":{"text":"A11034","term_id":"489250"}} A11034 ), Rabbit polyclonal anti-IL1 Receptor 1 (Abcam ab59995), isotype APC Mouse IgG 1 (BD pharmingen 555751), rabbit anti-mouse IL-1β (Abcam Ab9722), anti-CD14 (e-Bioscience 16-0149-82), anti-β-actin (Santa Cruz Biotechnology), Goat polyclonal anti-P-selectin (C-20; SC-6941), recombinant human Interleukin 1 Receptor Antagonist (rhIL1Ra) (R&D Systems).

    Article Title: Lipopolysaccharide stimulates platelets through an IL-1β autocrine loop
    Article Snippet: .. Chemicals and reagents Chemicals and reagents were purchased from the following sources: sterile filtered HBSS and M199 (BioWhittaker); sterile tissue culture plates, (Falcon Labware); human serum albumin, (Baxter Healthcare); endotoxin-free PBS, phenol-extracted LPS ( Escherichia coli O111:B4) that is free of lipoprotein contamination (List Biological Laboratories); recombinant soluble CD14, recombinant LBP, and IL-1β ELISA kits, (R&D Systems); Caspase-1 inhibitors z-WEHD-FMK (FMK002) and z-YVAD-FMK (FMK005) were obtained from R&D Systems. .. Alexa 594 -conjugated wheat germ agglutinin (Invitrogen), Alexa Flour 488 Goat anti rabbit (Invitrogen {"type":"entrez-nucleotide","attrs":{"text":"A11034","term_id":"489250"}} A11034 ), Rabbit polyclonal anti-IL1 Receptor 1 (Abcam ab59995), isotype APC Mouse IgG 1 (BD pharmingen 555751), rabbit anti-mouse IL-1β (Abcam Ab9722), anti-CD14 (e-Bioscience 16-0149-82), anti-β-actin (Santa Cruz Biotechnology), Goat polyclonal anti-P-selectin (C-20; SC-6941), recombinant human Interleukin 1 Receptor Antagonist (rhIL1Ra) (R&D Systems).

    Cell Attachment Assay:

    Article Title: Intestinal epithelium-derived high density lipoprotein restrains liver injury via the portal vein
    Article Snippet: The layer containing liver macrophages was plated in RPMI-1640 (Hyclone) containing 10% fetal bovine serum (FBS). .. After 2 h culture to allow for cell attachment, the cell medium was changed to “vehicle” medium, which was serum-free RPMI-1640 containing 1 μg/ml recombinant LBP (R&D systems; #6635-LP) for 3 h of culture. .. HDL preparations as described were added, or not, to these cultures with LPS (Sigma-Aldrich; #L2630) presence.

    Incubation:

    Article Title: Intestinal epithelium-derived high density lipoprotein restrains liver injury via the portal vein
    Article Snippet: For sandwich ELISA to analyze LPS-HDL binding, we used high-binding clear polystyrenes microplates (R&D systems, DY990), and purified HDL was immobilized for 2 h on these plates at 37°C at 10 μg/ml. .. After washing, plates were blocked with 1% BSA for 1 h. Then Biotin-LPS (InvivoGen; #tlrl-lpsbiot) was pre-incubated with or without 1 μg/ml recombinant LBP (R&D Systems) for 1 h and incubated in plates for 30 min. Streptavidin peroxidase (R&D Systems) was added, followed by diaminobenzidine substrate (Abcam), for colorimetric reactions. ..

    other:

    Article Title: Enterically derived high-density lipoprotein restrains liver injury through the portal vein.
    Article Snippet: Images were acquired with a transmission electron microscope (TEM; JEOL, #JEM-1400Plus) at 120 KeV and 80,000× or 150,000× magnifications.

    Article Title: Enterically derived high-density lipoprotein restrains liver injury through the portal vein.
    Article Snippet: ow nloaded from https://w w w .science.org on A pril 01, 2024 incubated with 100 ng/ml of biotinylated LPS (Invivogen, #tlrl-lpsbiot) for 2 hours.



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    A HUVECs were stimulated with ECVE nic or control (EBM-2) for 12 hours (incubation). Then, 100 ng/ml LPS with co-factors <t>LBP</t> and CD14 or co-factors alone (sham) were added and incubated for another 18 hours. Created in BioRender. Stahl, K. (2026) https://BioRender.com/nu4iwxz . B Angpt-2 concentrations in the supernatant were measured by ELISA when LPS was added as well as 12 and 18 hours after LPS or sham stimulation and are shown over time. Each group consisted of n =6. Mixed ANOVA with according post-tests were performed. C 18 hours after stimulation with LPS or sham Angpt-2 mRNA transcription was measured by qPCR. Each group consisted of n =9. D Additionally, the Angpt-2/Tie2 mRNA ratio is shown after initial ECVE nic incubation and 18 hours of LPS or sham stimulation. ECVE nic / sham consisted of n=8, all other groups consisted of n =9. Groups were analyzed by Kruskal-Wallis test with post-hoc Dunn test. E TER was measured at different time points by an ECIS system. HUVECs were pre-incubated with ECVE nic ( n =7) or control ( n =8) for 12 hours. Then half of the medium was changed and cells were stimulated with LPS. Results are shown as dot plots with mean ± SD. A linear mixed-effects model was used for analysis. P -values < 0.05 are considered significant. Abbreviations: Angpt-2 - Angiopoietin-2, ECIS - Electric cell-substrate impedance sensing, ECVE – e-cigarette vapor extract, HUVECs – human umbilical vein endothelial cells, LPS - <t>lipopolysaccharide,</t> SD – standard deviation, TER – transendothelial electrical resistance
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    92
    R&D Systems rlbp
    <t>LPS</t> binds to HIV-1 in a CD14-specific manner and does not passively associate with the lipid envelope. ( A ) Optimized LPS-bio (300 ng/mL, overnight, 4°C) and PE-SA (0.2 µg/mL, 4 h, 4°C) staining on CD14 + virions. Particle counts in PE + gates are enumerated on each plot. ( B ) Virion capture assay using immobilized untagged LPS. Ninety-six-well plates were coated with buffer (PBS) or LPS and virions with (CD14 + , orange bars) or without CD14 (WT, teal bars) were added. Captured virions were quantified by Gag p24 ELISA. Bars are mean ± SD of duplicate capture assays and are representative of three independent experiments. ( C ) FVM evaluating the contribution of recombinant LPS-binding protein (LBP) on LPS-bio binding to CD14 + virions. Varying amounts of recombinant LBP <t>(rLBP)</t> were mixed with LPS-bio prior to incubating with WT (top row) and CD14 + (bottom row) virus samples and staining with PE-SA. Gates define PE + events based on negative control (PE-SA only). WT and CD14 + virus stocks shown in FVM plots for ( A ) and ( C ) had a respective titer of 76 and 57 ng/mL p24 as measured by ELISA. ( D ) Particle counts in PE + gates from ( C ) were plotted as a function of PE-SA concentrations for WT (teal triangles) and CD14 + virions (orange circles). Data are mean ± SD of two independent virus stocks. ( E ) FVM measuring neutralization of LPS-bio binding to virions. Anti-CD14 (M5E2) was added to WT (top row) and CD14 + (bottom row) virions before incubation with LPS-bio and PE-SA. Gates are as described for ( C ). WT and CD14 + virus stocks shown in FVM plots had a respective titer of 368 and 141 ng/mL p24 as measured by ELISA. ( F ) Particle counts in PE + gates from ( E ) were plotted over the range of anti-CD14 concentrations tested for WT (teal triangles) and CD14 + (orange circles) virions. Data are representative of two independent virus stocks.
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    A HUVECs were stimulated with ECVE nic or control (EBM-2) for 12 hours (incubation). Then, 100 ng/ml LPS with co-factors LBP and CD14 or co-factors alone (sham) were added and incubated for another 18 hours. Created in BioRender. Stahl, K. (2026) https://BioRender.com/nu4iwxz . B Angpt-2 concentrations in the supernatant were measured by ELISA when LPS was added as well as 12 and 18 hours after LPS or sham stimulation and are shown over time. Each group consisted of n =6. Mixed ANOVA with according post-tests were performed. C 18 hours after stimulation with LPS or sham Angpt-2 mRNA transcription was measured by qPCR. Each group consisted of n =9. D Additionally, the Angpt-2/Tie2 mRNA ratio is shown after initial ECVE nic incubation and 18 hours of LPS or sham stimulation. ECVE nic / sham consisted of n=8, all other groups consisted of n =9. Groups were analyzed by Kruskal-Wallis test with post-hoc Dunn test. E TER was measured at different time points by an ECIS system. HUVECs were pre-incubated with ECVE nic ( n =7) or control ( n =8) for 12 hours. Then half of the medium was changed and cells were stimulated with LPS. Results are shown as dot plots with mean ± SD. A linear mixed-effects model was used for analysis. P -values < 0.05 are considered significant. Abbreviations: Angpt-2 - Angiopoietin-2, ECIS - Electric cell-substrate impedance sensing, ECVE – e-cigarette vapor extract, HUVECs – human umbilical vein endothelial cells, LPS - lipopolysaccharide, SD – standard deviation, TER – transendothelial electrical resistance

    Journal: Respiratory Research

    Article Title: The Tie-2 agonist PMC-403 inhibits endothelial damage induced by e-cigarette vapor or LPS

    doi: 10.1186/s12931-026-03928-4

    Figure Lengend Snippet: A HUVECs were stimulated with ECVE nic or control (EBM-2) for 12 hours (incubation). Then, 100 ng/ml LPS with co-factors LBP and CD14 or co-factors alone (sham) were added and incubated for another 18 hours. Created in BioRender. Stahl, K. (2026) https://BioRender.com/nu4iwxz . B Angpt-2 concentrations in the supernatant were measured by ELISA when LPS was added as well as 12 and 18 hours after LPS or sham stimulation and are shown over time. Each group consisted of n =6. Mixed ANOVA with according post-tests were performed. C 18 hours after stimulation with LPS or sham Angpt-2 mRNA transcription was measured by qPCR. Each group consisted of n =9. D Additionally, the Angpt-2/Tie2 mRNA ratio is shown after initial ECVE nic incubation and 18 hours of LPS or sham stimulation. ECVE nic / sham consisted of n=8, all other groups consisted of n =9. Groups were analyzed by Kruskal-Wallis test with post-hoc Dunn test. E TER was measured at different time points by an ECIS system. HUVECs were pre-incubated with ECVE nic ( n =7) or control ( n =8) for 12 hours. Then half of the medium was changed and cells were stimulated with LPS. Results are shown as dot plots with mean ± SD. A linear mixed-effects model was used for analysis. P -values < 0.05 are considered significant. Abbreviations: Angpt-2 - Angiopoietin-2, ECIS - Electric cell-substrate impedance sensing, ECVE – e-cigarette vapor extract, HUVECs – human umbilical vein endothelial cells, LPS - lipopolysaccharide, SD – standard deviation, TER – transendothelial electrical resistance

    Article Snippet: Unless specified otherwise, cells were stimulated with 100 ng/ml lipopolysaccharide (LPS) from E. coli serotype O111:B4 (L4130, Sigma-Aldrich, St. Louis, MO) or vehicle (EBM-2), 100 ng/ml lipopolysaccharide binding protein (LBP) (870-LP-025, R&D Systems, Minneapolis, MN), 100 ng/ml CD14 (SRP3149, Sigma–Aldrich, St. Louis, MO), 500 ng/ml Angpt-1 (923-AN/CF, R&D Systems, Minneapolis, MN), electronic cigarette vapor extract (ECVE, see below) and/or different concentrations (e.g. 0.3, 1, 3–10 μg/ml) of the Tie2 agonist PMC-403 (PharmAbcine, Daejeon, Republic of Korea).

    Techniques: Control, Incubation, Enzyme-linked Immunosorbent Assay, Analysis, Electric Cell-substrate Impedance Sensing, Standard Deviation

    LPS binds to HIV-1 in a CD14-specific manner and does not passively associate with the lipid envelope. ( A ) Optimized LPS-bio (300 ng/mL, overnight, 4°C) and PE-SA (0.2 µg/mL, 4 h, 4°C) staining on CD14 + virions. Particle counts in PE + gates are enumerated on each plot. ( B ) Virion capture assay using immobilized untagged LPS. Ninety-six-well plates were coated with buffer (PBS) or LPS and virions with (CD14 + , orange bars) or without CD14 (WT, teal bars) were added. Captured virions were quantified by Gag p24 ELISA. Bars are mean ± SD of duplicate capture assays and are representative of three independent experiments. ( C ) FVM evaluating the contribution of recombinant LPS-binding protein (LBP) on LPS-bio binding to CD14 + virions. Varying amounts of recombinant LBP (rLBP) were mixed with LPS-bio prior to incubating with WT (top row) and CD14 + (bottom row) virus samples and staining with PE-SA. Gates define PE + events based on negative control (PE-SA only). WT and CD14 + virus stocks shown in FVM plots for ( A ) and ( C ) had a respective titer of 76 and 57 ng/mL p24 as measured by ELISA. ( D ) Particle counts in PE + gates from ( C ) were plotted as a function of PE-SA concentrations for WT (teal triangles) and CD14 + virions (orange circles). Data are mean ± SD of two independent virus stocks. ( E ) FVM measuring neutralization of LPS-bio binding to virions. Anti-CD14 (M5E2) was added to WT (top row) and CD14 + (bottom row) virions before incubation with LPS-bio and PE-SA. Gates are as described for ( C ). WT and CD14 + virus stocks shown in FVM plots had a respective titer of 368 and 141 ng/mL p24 as measured by ELISA. ( F ) Particle counts in PE + gates from ( E ) were plotted over the range of anti-CD14 concentrations tested for WT (teal triangles) and CD14 + (orange circles) virions. Data are representative of two independent virus stocks.

    Journal: Journal of Virology

    Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

    doi: 10.1128/jvi.00363-24

    Figure Lengend Snippet: LPS binds to HIV-1 in a CD14-specific manner and does not passively associate with the lipid envelope. ( A ) Optimized LPS-bio (300 ng/mL, overnight, 4°C) and PE-SA (0.2 µg/mL, 4 h, 4°C) staining on CD14 + virions. Particle counts in PE + gates are enumerated on each plot. ( B ) Virion capture assay using immobilized untagged LPS. Ninety-six-well plates were coated with buffer (PBS) or LPS and virions with (CD14 + , orange bars) or without CD14 (WT, teal bars) were added. Captured virions were quantified by Gag p24 ELISA. Bars are mean ± SD of duplicate capture assays and are representative of three independent experiments. ( C ) FVM evaluating the contribution of recombinant LPS-binding protein (LBP) on LPS-bio binding to CD14 + virions. Varying amounts of recombinant LBP (rLBP) were mixed with LPS-bio prior to incubating with WT (top row) and CD14 + (bottom row) virus samples and staining with PE-SA. Gates define PE + events based on negative control (PE-SA only). WT and CD14 + virus stocks shown in FVM plots for ( A ) and ( C ) had a respective titer of 76 and 57 ng/mL p24 as measured by ELISA. ( D ) Particle counts in PE + gates from ( C ) were plotted as a function of PE-SA concentrations for WT (teal triangles) and CD14 + virions (orange circles). Data are mean ± SD of two independent virus stocks. ( E ) FVM measuring neutralization of LPS-bio binding to virions. Anti-CD14 (M5E2) was added to WT (top row) and CD14 + (bottom row) virions before incubation with LPS-bio and PE-SA. Gates are as described for ( C ). WT and CD14 + virus stocks shown in FVM plots had a respective titer of 368 and 141 ng/mL p24 as measured by ELISA. ( F ) Particle counts in PE + gates from ( E ) were plotted over the range of anti-CD14 concentrations tested for WT (teal triangles) and CD14 + (orange circles) virions. Data are representative of two independent virus stocks.

    Article Snippet: Where indicated, LPS-bio was pre-incubated with increasing concentrations of rLBP (1 μL; R&D Systems #870-LP-025) or purified anti-CD14 (1 μL; mAb M5E2, BD Biosciences #557152) before adding to samples overnight and subsequently developing with PE-SA the next day.

    Techniques: Staining, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Virus, Negative Control, Neutralization, Incubation