Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Basal TLR4 expression by (A) primary adult baboon lung DCs and (B) KG-1-derived DCs under steady-state condition. Cell-surface expression of TLR4 was detected by flow cytometry after staining the cells with TLR4-specific antibody. The percent number and MFI values of cells stained with TLR4-specific antibody (TLR4) are compared with isotype control antibody-stained cells (I) in M1 region. (C): Western blot showing undetectable expression of TLR4 in 5 μg cell lysate protein of KG-1-derived DCs (KG1-DC). An equal amount of cell lysate protein of HEK293 cells stably transfected with TLR4 (HEK-TLR4) served as positive control.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Staining, Western Blot, Stable Transfection, Transfection, Positive Control

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Localization of exogenously-added recombinant TLR4-MD2 protein by confocal microscopy and flow-cytometry. Confocal microscopic images of KG-1-derived DCs pulsed with Alexa-fluor 594-conjugated recombinant TLR4-MD2 protein for (A) 1h and (B) 4h. Vybrant DiO (green) dye stains the cytoplasm, and Hoechst 33342 (blue) dye stains the nucleus of the cell. The images were acquired using 63 X objective. (C) Flow-cytometric charts of KG-1-derived DCs pulsed with Alexa-fluor 495-conjugated recombinant TLR4-MD2 protein after 1h (dark line) and 4h (dotted line). The histogram chart of non-pulsed cells (negative control) is shown under the black area. Cells were gated in M region. Percent number of cells (and MFI values) positive for fluorescence are shown within the chart. Results are representative of two experiments.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Recombinant, Confocal Microscopy, Flow Cytometry, Derivative Assay, Negative Control, Fluorescence

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A, recombinant TLR4-MD2 protein and MD2 protein on phagocytic function of KG-1-derived DCs. (A): Confocal microscopic images of KG-1-derived DCs incubated with pHrodo-labeled E. coli bioparticles for 3h. Phagocytosed bioparticles fluoresce red. Cells without phagocytosed particles and extracellular bacteria do not fluoresce. Enlarged images of a cell (shown as circle) are also shown in the figure, at different z-stack slices. (B): The extracellular bacteria that are either settled at the bottom or lie towards the top do not emit any fluorescence. These images confirm that fluorescence is of phagocytosed bioparticles. Next, KG-1-derived DCs were incubated with (C): purified baboon lung SP-A (0.2 and 2 μM), (D): recombinant TLR4-MD2 protein (0.06–0.6 μM) and functional-grade anti-human TLR4 antibody (HTA125 clone, Imgenex, CA; control reaction), (E): recombinant MD2 protein (0.02–0.2 μM), and (F): purified baboon lung SP-A (2 μM) and TLR4-MD2 protein (0.6 μM), for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically at 550 nm excitation and 600 nm emission wavelengths. Results are mean (SEM) of three different experiments. * p<0.05 or ns: not significant as compared to basal phagocytosis.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Derivative Assay, Incubation, Labeling, Bacteria, Fluorescence, Functional Assay

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of simultaneous addition of purified SP-A and recombinant TLR4-MD2 protein on phagocytic function of primary (A) adult baboon lung DCs and (B) fetal baboon lung DC-precursor cells. The DCs were incubated with respective proteins for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically. * p<0.05, ns: not significant or otherwise indicated. Results are mean (SEM) of three different experiments performed at different times.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A and recombinant TLR4-MD2 proteins on TNF-α secretion by DCs against E. coli. (A) Primary adult baboon lung DCs or (B) fetal baboon lung DC-precursor cells were incubated with effector molecules for an hour prior to addition of pHrodo-labeled E. coli bioparticles. After 3h incubation at 37°C in 5% CO2 incubator, the cell-free supernatants were collected and subjected to ELISA for measurement of TNF-α. The results are representative of two experiments performed separately in triplicate. * p<0.05, ** p<0.001, ns: not significant.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Basal TLR4 expression by (A) primary adult baboon lung DCs and (B) KG-1-derived DCs under steady-state condition. Cell-surface expression of TLR4 was detected by flow cytometry after staining the cells with TLR4-specific antibody. The percent number and MFI values of cells stained with TLR4-specific antibody (TLR4) are compared with isotype control antibody-stained cells (I) in M1 region. (C): Western blot showing undetectable expression of TLR4 in 5 μg cell lysate protein of KG-1-derived DCs (KG1-DC). An equal amount of cell lysate protein of HEK293 cells stably transfected with TLR4 (HEK-TLR4) served as positive control.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Expressing, Derivative Assay, Flow Cytometry, Staining, Western Blot, Stable Transfection, Transfection, Positive Control

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Localization of exogenously-added recombinant TLR4-MD2 protein by confocal microscopy and flow-cytometry. Confocal microscopic images of KG-1-derived DCs pulsed with Alexa-fluor 594-conjugated recombinant TLR4-MD2 protein for (A) 1h and (B) 4h. Vybrant DiO (green) dye stains the cytoplasm, and Hoechst 33342 (blue) dye stains the nucleus of the cell. The images were acquired using 63 X objective. (C) Flow-cytometric charts of KG-1-derived DCs pulsed with Alexa-fluor 495-conjugated recombinant TLR4-MD2 protein after 1h (dark line) and 4h (dotted line). The histogram chart of non-pulsed cells (negative control) is shown under the black area. Cells were gated in M region. Percent number of cells (and MFI values) positive for fluorescence are shown within the chart. Results are representative of two experiments.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Recombinant, Confocal Microscopy, Flow Cytometry, Derivative Assay, Negative Control, Fluorescence

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A, recombinant TLR4-MD2 protein and MD2 protein on phagocytic function of KG-1-derived DCs. (A): Confocal microscopic images of KG-1-derived DCs incubated with pHrodo-labeled E. coli bioparticles for 3h. Phagocytosed bioparticles fluoresce red. Cells without phagocytosed particles and extracellular bacteria do not fluoresce. Enlarged images of a cell (shown as circle) are also shown in the figure, at different z-stack slices. (B): The extracellular bacteria that are either settled at the bottom or lie towards the top do not emit any fluorescence. These images confirm that fluorescence is of phagocytosed bioparticles. Next, KG-1-derived DCs were incubated with (C): purified baboon lung SP-A (0.2 and 2 μM), (D): recombinant TLR4-MD2 protein (0.06–0.6 μM) and functional-grade anti-human TLR4 antibody (HTA125 clone, Imgenex, CA; control reaction), (E): recombinant MD2 protein (0.02–0.2 μM), and (F): purified baboon lung SP-A (2 μM) and TLR4-MD2 protein (0.6 μM), for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically at 550 nm excitation and 600 nm emission wavelengths. Results are mean (SEM) of three different experiments. * p<0.05 or ns: not significant as compared to basal phagocytosis.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Purification, Recombinant, Derivative Assay, Incubation, Labeling, Fluorescence, Functional Assay

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of simultaneous addition of purified SP-A and recombinant TLR4-MD2 protein on phagocytic function of primary (A) adult baboon lung DCs and (B) fetal baboon lung DC-precursor cells. The DCs were incubated with respective proteins for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically. * p<0.05, ns: not significant or otherwise indicated. Results are mean (SEM) of three different experiments performed at different times.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Purification, Recombinant, Incubation, Labeling

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A and recombinant TLR4-MD2 proteins on TNF-α secretion by DCs against E. coli. (A) Primary adult baboon lung DCs or (B) fetal baboon lung DC-precursor cells were incubated with effector molecules for an hour prior to addition of pHrodo-labeled E. coli bioparticles. After 3h incubation at 37°C in 5% CO2 incubator, the cell-free supernatants were collected and subjected to ELISA for measurement of TNF-α. The results are representative of two experiments performed separately in triplicate. * p<0.05, ** p<0.001, ns: not significant.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Purification, Recombinant, Incubation, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine

Article Title: Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance

doi: 10.1186/s10020-018-0023-8

Figure Lengend Snippet: SPR analyses of redox forms of HMGB1 binding to TLR4/MD-2 complex or TLR4. a - c TLR4/MD-2 complex was coated on the CM5 chip; disulfide HMGB1 binds to complex with a K D of 0.42 ± 0.01 μM; reduced HMGB1 binds with a K D of 3.93 ± 0.01 μM; HMGB1 3S mutant binds with a K D of 3.02 ± 0.02 μM. d - f TLR4 was coated on the chip; HMGB1 binds to TLR4 with a K D of 0.64 ± 0.01 μM; reduced HMGB1 binds with a K D of 0.65 ± 0.01 μM; HMGB1 3S mutant binds with a K D of 4.20 ± 0.09 μM. Data are representative of three repeats

Article Snippet: Human TLR4/MD-2 complex, human MD-2, TLR4 were obtained from R&D Systems.

Techniques: Binding Assay, Mutagenesis

Journal: Molecular Medicine

Article Title: Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance

doi: 10.1186/s10020-018-0023-8

Figure Lengend Snippet: SPR analyses of GST-A-box binding to TLR4/MD-2, TLR4 and MD-2. a TLR4/MD-2 complex was coated on the CM5 chip, GST-A-box binds to complex with a K D of 1.21 ± 0.02 μM. b TLR4 was coated on the chip, GST-A-box binds to TLR4 with a K D of 0.59 ± 0.01 μM. c GST-A-box was coated on the CM5 chips, MD-2 was used as analyte, MD-2 binds to GST-A-box with a K D of 3.25 ± 0.17 μM. GST tag was used as negative control. Data are representative of three repeats

Article Snippet: Human TLR4/MD-2 complex, human MD-2, TLR4 were obtained from R&D Systems.

Techniques: Binding Assay, Negative Control

Journal: Molecular Medicine

Article Title: Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance

doi: 10.1186/s10020-018-0023-8

Figure Lengend Snippet: SPR analyses of GST-B-box binding to TLR4/MD-2, TLR4 and MD-2. a TLR4/MD-2 complex was coated on the CM5 chip, GST-B-box binds to TLR4/MD-2 complex with a K D of 5.78 ± 0.22 μM. b TLR4 was coated on the chip, GST-B-box has no significant binding to TLR4. c GST-B-box was coated on the CM5 chips, MD-2 was used as analyte, MD-2 showed better binding affinity to GST-B-box (K D of 1.41 ± 0.03 μM) relative to GST-A-box (K D = 3.25 ± 0.17 μM). GST tag was used as negative control. Data are representative of three repeats

Article Snippet: Human TLR4/MD-2 complex, human MD-2, TLR4 were obtained from R&D Systems.

Techniques: Binding Assay, Negative Control

Journal: Molecular Medicine

Article Title: Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance

doi: 10.1186/s10020-018-0023-8

Figure Lengend Snippet: Ternary complex formation among TLR4/MD-2, TLR4, A-box and HMGB1. a Biacore sensorgram showing molecular interactions, after injecting HMGB1 (100 nM) followed immediately by HBS buffer, HMGB1 (100 nM) or TLR4/MD-2 (100 nM) or mixture of the latter (100 nM each) onto a TLR4/MD-2 sensor chip surface. b Injecting A-box (10 μM), followed immediately by A-box (0 μg/ml of HMGB1) or HMGB1 (2.5 and 5 μg/ml) onto a TLR4 sensor chip surface

Article Snippet: Human TLR4/MD-2 complex, human MD-2, TLR4 were obtained from R&D Systems.

Techniques:

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: HSP72 and HSP105 on the surface of TEXs mediated TLR2- and TLR4-dependent IL-6 secretion of DCs. (A) BMDCs were pre-incubated with anti-TLR2, TLR4 or both mAbs at a concentration of 30 μg/ml for 1 h, and BMDCs were stimulated with 5 μg/ml B16-F10-EXO for 6 h. IL-6 production from BMDCs was measured by ELISA. (B, C) Exosomes from B16-F10 cells transfected with HSP72, HSP105 or HSC70 siRNA, or NC siRNA were isolated. The HSP protein knockdown effect was detected by Western blot (B). BMDCs were stimulated with 5 μg/ml of the indicated B16-F10-EXO for 6 h, and IL-6 production from BMDCs was measured by ELISA (C). (D, E) After adsorption onto latex beads (D) or anti-CD63-coated latex beads (E), HSP72 and HSP105 on exosomes were detected by flow cytometry. (F, G) BMDCs were pretreated with 2.5 μg/ml cytochalasin D for 30 min, and co-cultured with CFSE-labeled B16-F10-EXO for 6 h. The uptake of B16-F10-EXO by BMDCs was detected by confocal microscopy (F), or stimulated with B16-F10-EXO for 6 h. IL-6 production by BMDCs was measured by ELISA (G). (B) Numbers indicate the ratio of gray values of the corresponding protein to that of CD63. (A, C, G) The results are shown as the mean ± SEM of 3 independent experiments (n = 3). (B, D, E, F) One representative of 3 independent experiments is shown. P values were generated by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test; *p < 0.05 and ***p< 0.001 versus B16-F10-EXO in A.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Isolation, Western Blot, Adsorption, Flow Cytometry, Cell Culture, Labeling, Confocal Microscopy, Generated

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: HSP105 acts as a ligand of TLR2 and TLR4 to induce DC IL-6 production. (A) Affinity precipitation of recombinant His-TLR2 or His-TLR4 protein with Flag-EGFP or Flag-HSP105 protein, captured by anti-FLAG® M2 magnetic beads and analyzed by immunoblot analysis with Abs against TLR2 and TLR4. (B) BMDCs were pre-incubated with anti-TLR2, TLR4 or both mAbs at a concentration of 30 μg/ml for 1 h, and then stimulated with 2 μg/ml endotoxin-free Flag-EGFP or Flag-HSP105 protein for 6 h. IL-6 production was measured by ELISA (n = 3). (A) One representative of 3 independent experiments is shown. (B) The results are shown as the mean ± SEM of 3 independent experiments. P values were generated by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: Affinity Precipitation, Recombinant, Magnetic Beads, Western Blot, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Generated

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Basal TLR4 expression by (A) primary adult baboon lung DCs and (B) KG-1-derived DCs under steady-state condition. Cell-surface expression of TLR4 was detected by flow cytometry after staining the cells with TLR4-specific antibody. The percent number and MFI values of cells stained with TLR4-specific antibody (TLR4) are compared with isotype control antibody-stained cells (I) in M1 region. (C): Western blot showing undetectable expression of TLR4 in 5 μg cell lysate protein of KG-1-derived DCs (KG1-DC). An equal amount of cell lysate protein of HEK293 cells stably transfected with TLR4 (HEK-TLR4) served as positive control.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Expressing, Derivative Assay, Flow Cytometry, Staining, Western Blot, Stable Transfection, Transfection, Positive Control

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Localization of exogenously-added recombinant TLR4-MD2 protein by confocal microscopy and flow-cytometry. Confocal microscopic images of KG-1-derived DCs pulsed with Alexa-fluor 594-conjugated recombinant TLR4-MD2 protein for (A) 1h and (B) 4h. Vybrant DiO (green) dye stains the cytoplasm, and Hoechst 33342 (blue) dye stains the nucleus of the cell. The images were acquired using 63 X objective. (C) Flow-cytometric charts of KG-1-derived DCs pulsed with Alexa-fluor 495-conjugated recombinant TLR4-MD2 protein after 1h (dark line) and 4h (dotted line). The histogram chart of non-pulsed cells (negative control) is shown under the black area. Cells were gated in M region. Percent number of cells (and MFI values) positive for fluorescence are shown within the chart. Results are representative of two experiments.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Recombinant, Confocal Microscopy, Flow Cytometry, Derivative Assay, Negative Control, Fluorescence

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A, recombinant TLR4-MD2 protein and MD2 protein on phagocytic function of KG-1-derived DCs. (A): Confocal microscopic images of KG-1-derived DCs incubated with pHrodo-labeled E. coli bioparticles for 3h. Phagocytosed bioparticles fluoresce red. Cells without phagocytosed particles and extracellular bacteria do not fluoresce. Enlarged images of a cell (shown as circle) are also shown in the figure, at different z-stack slices. (B): The extracellular bacteria that are either settled at the bottom or lie towards the top do not emit any fluorescence. These images confirm that fluorescence is of phagocytosed bioparticles. Next, KG-1-derived DCs were incubated with (C): purified baboon lung SP-A (0.2 and 2 μM), (D): recombinant TLR4-MD2 protein (0.06–0.6 μM) and functional-grade anti-human TLR4 antibody (HTA125 clone, Imgenex, CA; control reaction), (E): recombinant MD2 protein (0.02–0.2 μM), and (F): purified baboon lung SP-A (2 μM) and TLR4-MD2 protein (0.6 μM), for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically at 550 nm excitation and 600 nm emission wavelengths. Results are mean (SEM) of three different experiments. * p<0.05 or ns: not significant as compared to basal phagocytosis.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Purification, Recombinant, Derivative Assay, Incubation, Labeling, Fluorescence, Functional Assay

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of simultaneous addition of purified SP-A and recombinant TLR4-MD2 protein on phagocytic function of primary (A) adult baboon lung DCs and (B) fetal baboon lung DC-precursor cells. The DCs were incubated with respective proteins for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically. * p<0.05, ns: not significant or otherwise indicated. Results are mean (SEM) of three different experiments performed at different times.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Purification, Recombinant, Incubation, Labeling

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A and recombinant TLR4-MD2 proteins on TNF-α secretion by DCs against E. coli. (A) Primary adult baboon lung DCs or (B) fetal baboon lung DC-precursor cells were incubated with effector molecules for an hour prior to addition of pHrodo-labeled E. coli bioparticles. After 3h incubation at 37°C in 5% CO2 incubator, the cell-free supernatants were collected and subjected to ELISA for measurement of TNF-α. The results are representative of two experiments performed separately in triplicate. * p<0.05, ** p<0.001, ns: not significant.

Article Snippet: Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer’s directions.

Techniques: Purification, Recombinant, Incubation, Labeling, Enzyme-linked Immunosorbent Assay