Journal:

Article Title: Phospholipase D2 Is Localized to the Rims of the Golgi Apparatus in Mammalian Cells

doi: 10.1091/mbc.02-04-0059

Figure Lengend Snippet: PLD2 localization with different organelles in rat NRK cells. Cells were prepared for immunofluorescence microscopy and incubated with rabbit PLD2 antibody PLD2-27 (A, D, G, and J) and costained with monoclonal antibodies to the ER marker BiP (B); the transferrin receptor (E), a marker of early endosomes and the plasma membrane; and lgp120 (H), a late endosome/lysosomal marker. (K) Cells were stained with caveolin-1, a marker for caveoli; the arrow corresponds to caveolin-1 localized to the plasma membrane. Images were merged to determine overlap between PLD2 (red) and the respective marker proteins (green; C, F, I, and L). Areas of maximal overlap are yellow. (L) Inset, enlargement of the region of overlap between PLD2 and caveolin-1 in the perinuclear Golgi apparatus. The arrow indicates areas where PLD2 puncta (red) either overlap completely with caveolin-1 (green) or are directly adjacent. Images are from projected Z-series. Bars, 10 μm.

Article Snippet: Purified monoclonal anti-rat transferrin receptor antibodies were purchased from Cedarlane Laboratories (Hornby, Ontario, Canada).

Techniques: Immunofluorescence, Microscopy, Incubation, Marker, Staining

Journal:

Article Title: Survival of Aspergillus fumigatus in Serum Involves Removal of Iron from Transferrin: the Role of Siderophores

doi: 10.1128/IAI.72.3.1402-1408.2004

Figure Lengend Snippet: Degradation of transferrin by A. fumigatus in liquid cultures. A. fumigatus was incubated in MEM containing 10% human serum (A) or 2.5 μM holotransferrin (B). Supernatants were withdrawn from the cultures after the number of hours indicated above the lanes, and the presence of transferrin was determined by Western blotting following sodium dodecyl sulfate-PAGE. Controls (lanes C) were uninoculated samples incubated for 286 h. The band underneath transferrin in panel A is another protein that cross-reacted with the polyclonal antitransferrin antibody.

Article Snippet: Gels were silver stained or transferred to polyvinylidene difluoride membranes (Bio-Rad), blocked with 5% bovine serum albumin, probed with a rabbit immunoglobulin G fraction of anti-human transferrin (1:1,000 dilution; Rockland Inc., Gilbertsville, Pa.), and treated with goat anti-rabbit horseradish peroxidase.

Techniques: Incubation, Western Blot

Journal:

Article Title: Survival of Aspergillus fumigatus in Serum Involves Removal of Iron from Transferrin: the Role of Siderophores

doi: 10.1128/IAI.72.3.1402-1408.2004

Figure Lengend Snippet: Iron removal from transferrin by A. fumigatus. A. fumigatus was cultured in MEM containing 2.5 μM purified human holotransferrin (A) or 10% human serum (B). Culture media were withdrawn, and the iron saturation of transferrin was analyzed by urea-PAGE. Transferrin was visualized by Western blotting. The numbers above the lanes represent the hours of incubation with A. fumigatus. Fe2-Tf, holotransferrin; Apo-Tf, apotransferrin.

Article Snippet: Gels were silver stained or transferred to polyvinylidene difluoride membranes (Bio-Rad), blocked with 5% bovine serum albumin, probed with a rabbit immunoglobulin G fraction of anti-human transferrin (1:1,000 dilution; Rockland Inc., Gilbertsville, Pa.), and treated with goat anti-rabbit horseradish peroxidase.

Techniques: Cell Culture, Purification, Western Blot, Incubation

Journal:

Article Title: Survival of Aspergillus fumigatus in Serum Involves Removal of Iron from Transferrin: the Role of Siderophores

doi: 10.1128/IAI.72.3.1402-1408.2004

Figure Lengend Snippet: A. fumigatus can transport iron from transferrin across a dialysis membrane. A. fumigatus was inoculated into MEM in which a dialysis bag containing holotransferrin (25 μM) was suspended. A. fumigatus was incubated for 48 h, and then transferrin was withdrawn from the dialysis bag and analyzed by urea-PAGE (lane +). An uninoculated control flask containing MEM plus transferrin in a dialysis bag also was examined (lane −). Pure holotransferrin (Fe2-Tf) and apotransferrin (Apo-Tf) standards also were run.

Article Snippet: Gels were silver stained or transferred to polyvinylidene difluoride membranes (Bio-Rad), blocked with 5% bovine serum albumin, probed with a rabbit immunoglobulin G fraction of anti-human transferrin (1:1,000 dilution; Rockland Inc., Gilbertsville, Pa.), and treated with goat anti-rabbit horseradish peroxidase.

Techniques: Incubation

Journal:

Article Title: Survival of Aspergillus fumigatus in Serum Involves Removal of Iron from Transferrin: the Role of Siderophores

doi: 10.1128/IAI.72.3.1402-1408.2004

Figure Lengend Snippet: Iron saturation of transferrin following incubation with A. fumigatus siderophores. Purified desferrisiderophores were incubated with holotransferrin (25 μM) at 37°C for 16 h. Desferriferrichrome, desferriferricrocin, and desferritriacetylfusarinine C were serially diluted to final concentrations of 5 μM (lanes 1), 50 μM (lanes 2), 500 μM (lanes 3), and 5 mM (lanes 4). Controls containing holotransferrin alone also were run (lanes 0). holo-Tf, holotransferrin; Fe-Tf, monoferric transferrin; apo-Tf, apotransferrin.

Article Snippet: Gels were silver stained or transferred to polyvinylidene difluoride membranes (Bio-Rad), blocked with 5% bovine serum albumin, probed with a rabbit immunoglobulin G fraction of anti-human transferrin (1:1,000 dilution; Rockland Inc., Gilbertsville, Pa.), and treated with goat anti-rabbit horseradish peroxidase.

Techniques: Incubation, Purification

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

doi: 10.1016/j.biopha.2024.117723

Figure Lengend Snippet: Fig. 5. In vivo HU treatment of Nos2–/– mice impairs HU inhibition of proliferation in erythroid progenitors. a) Schematic representation of experimental setup: Nos2–/– or wild-type (WT) mice were treated orally with 200 mg/kg HU or drinking water for 2 weeks. WT mice were injected with 20 mg/kg of 1400W twice daily for 3 consecutive days. Mouse erythroid progenitors (mERP) were isolated from bone marrow by immunomagnetic cell separation using anti-CD71-PE and anti- Ter119-FITC antibodies. b) Immunocytochemistry for Nos2 protein in mERP isolated from WT mice treated or not with HU. Quantification of Nos2-positive cells. c) Citrulline concentration in the bone marrow of WT and Nos2–/– mice treated or not with HU. d) Colony formation assay showing the number of late erythroid (CFU-E), early erythroid (BFU-E), or granulocyte/macrophage progenitors (CFU-GM) in the bone marrow of WT or Nos2–/– mice treated or not with HU. e) Immunocytochemistry for Ki67 in mERP cells isolated from WT and Nos2–/– mice treated or not with HU. f) Quantification of Ki67-positive cells. g) Cell cycle distribution by flow cytometry showing the percentage of cells in G0/G1, S, or G2/M phases of the cell cycle. c) n = 3, f) n = 5; mean + SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT.

Article Snippet: Bone marrow cells were flushed out of the femurs and tibias of WT or Nos2–/– mice treated with HU or 1400W and resuspended in 100 μL of buffer (1 % PBS, 0.5 % BSA, 2 mM EDTA) and stained with 7 μL of anti-human CD71-PE REAfinityTM antibody (Miltenyi Biotec, 130–120–809, Bergisch Gladbach, Germany) for 15 min at 4◦C.

Techniques: In Vivo, Inhibition, Injection, Isolation, Immunocytochemistry, Concentration Assay, Colony Assay, Flow Cytometry

Journal: Theranostics

Article Title: Determination of cerebrospinal fluid leakage by selective deletion of transferrin glycoform using an immunochromatographic assay

doi: 10.7150/thno.34411

Figure Lengend Snippet: Schematic illustration of detection strategy of beta-2 transferrin (β2TF) for determination of cerebrospinal fluid (CSF) leakage. ( A ) Sample pretreatment process, ( B ) structure of immunochromatographic assay (ICA), and ( C ) reaction mechanisms of the entire process.

Article Snippet: Anti-transferrin monoclonal antibody (4T15-8B9; conjugated Ab, 4T15-11D3; immobilized Ab) was obtained from Hytest (Joukahaisenkatu, Turku, Finland).

Techniques: Immunochromatographic Assay

Journal: Theranostics

Article Title: Determination of cerebrospinal fluid leakage by selective deletion of transferrin glycoform using an immunochromatographic assay

doi: 10.7150/thno.34411

Figure Lengend Snippet: Demonstration of specificity of lectin in immunochromatographic assay (ICA) by evaluation of ( A ) oxidation effect of antibody. ( B ) Comparison of colorimetric signal intensity between sialo-transferrin (sTF), beta-2 transferrin (β2TF), and control.

Article Snippet: Anti-transferrin monoclonal antibody (4T15-8B9; conjugated Ab, 4T15-11D3; immobilized Ab) was obtained from Hytest (Joukahaisenkatu, Turku, Finland).

Techniques: Immunochromatographic Assay, Comparison, Control

Journal: Biology of reproduction

Article Title: Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells.

doi: 10.1095/biolreprod.107.063107

Figure Lengend Snippet: FIG. 1. Purification and functional char- acterization of dimeric TRF. A) Chromato- graphic profile of highly purified human TRF. The TRF solution was loaded onto an ultragel Aca44 column as described in Materials and Methods. OD, optical density. B) Each fraction was analyzed using native gel electrophoresis and immunoblotting. VLMB, very low-mobility band; LMB, low- mobility band; HMB, high-mobility band. C) Sensorgrams of interaction between high- and low-mobility purified fractions of hTRF and the monoclonal anti-hTRF anti- body. The interaction between both forms of hTRF and the monoclonal antibody is indicated by a dashed line. After reinjection of the monoclonal antibody, only the accessible epitope on the dimeric form generated a secondary signal (dark line), in contrast to the monomeric form (light line). The arrows directed to the top indicate the beginning of injection of the anti-hTRF antibody, and those directed toward the bottom indicate the end of the injections. The principle of discrimination between monomeric versus dimeric TRF by SPR is schematized on the right. RU, resonance units.

Article Snippet: Monoclonal mouse antibodies raised against the N-terminal domain of human TRF were from AbD Serotec (Cergy Saint-Christophe, France).

Techniques: Purification, Functional Assay, Nucleic Acid Electrophoresis, Western Blot, Generated, Injection

Journal: Biology of reproduction

Article Title: Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells.

doi: 10.1095/biolreprod.107.063107

Figure Lengend Snippet: FIG. 5. Production of monomeric and dimeric forms of TRF by Sertoli cells. Western blots in nondenaturating conditions were carried out to analyze TRF in cell media of Sertoli cells stimulated by FSH (100 ng/ml) and by insulin (100 ng/ml) for 24 h (lane 2) and in lysates (lanes 4 and 5). Lane 1: human polymers of TRF fractionated by gel filtration (see Materials and Methods) ; lane 2: Sertoli cell culture medium ; lane 3: purified human dimeric TRF ; lane 4: Sertoli cell lysate ; lane 5: Sertoli cell lysate (increased contrast to visualize the intracellular monomer in lane 4). Lanes 1 and 3 were hybridized with an anti-human TRF antibody, and lanes 2 and 4 were hybridized with an anti-rat TRF antibody. Samples of lanes 1 and 2 migrated in one gel. Likewise, samples corresponding to lanes 3 and 4 migrated in another gel.

Article Snippet: Monoclonal mouse antibodies raised against the N-terminal domain of human TRF were from AbD Serotec (Cergy Saint-Christophe, France).

Techniques: Western Blot, Filtration, Cell Culture, Purification

Journal:

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/IAI.72.6.3204-3217.2004

Figure Lengend Snippet: Live and formalin-killed F. tularensis bacteria exhibit similar kinetics of colocalization with the early endosomal marker EEA1. (A) Preopsonized formalin-killed (a to c) or live F. tularensis RCI (d and e) was incubated with THP-1 cells at 0°C to allow adherence, but not uptake, washed, warmed to 37°C to allow for internalization, fixed after 10 to 60 min, and stained by immunofluorescence for EEA1 (green) (a and d) and for F. tularensis antigen (red) (b and e). Panels c and f show the merged green and red images. The panels show THP-1 cells fixed after 15 min. Arrows indicate bacteria that colocalized with EEA1. (B) Percentages of live (RCI or LVS) or formalin-killed (FK) F. tularensis RCI bacteria colocalizing with EEA1. The data represent the means ± standard errors. The experiment was performed three times, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Marker, Incubation, Staining, Immunofluorescence

Journal:

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/IAI.72.6.3204-3217.2004

Figure Lengend Snippet: Virulent RCI of F. tularensis colocalizes extensively with LAMP CD63 at 4 h but very little at 16 h after uptake by human THP-1 cells. THP-1 cells were infected with the RCI of F. tularensis, fixed 4 or 16 h after infection, stained for CD63 (Oregon Green), F. tularensis (Texas Red), and DNA (DAPI), and imaged by laser scanning confocal and two-photon fluorescence microscopy. After 4 h (top row), the majority of F. tularensis bacteria resided in compartments with intense staining around the rim for CD63 (arrows). After 16 h (bottom row), the bacteria had multiplied extensively and no longer colocalized with CD63. This experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Infection, Staining, Fluorescence, Microscopy

Journal:

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/IAI.72.6.3204-3217.2004

Figure Lengend Snippet: Quantitation of immunofluorescence staining for CD63 in THP-1 cells infected with F. tularensis RCI or LVS. Monolayers of human THP-1 cells were incubated with latex beads, formalin-killed F. tularensis, or live F. tularensis LVS or RCI as described in the text. Monolayers were washed, incubated for an additional 1 to 16 h, fixed, permeabilized, stained for CD63 and F. tularensis by using immunofluorescence reagents, and examined by confocal and two-photon laser scanning fluorescence microscopy, and the numbers of bacteria and latex beads that colocalized with CD63 were enumerated. Colocalization of the F. tularensis RCI and LVS with CD63 reached a maximum of 60 to 70% 2 to 4 h after infection. Thereafter, with continued intracellular multiplication, the colocalization of F. tularensis with CD63 steadily declined, falling to 15% by 16 h. Latex beads and formalin-killed F. tularensis colocalized extensively (70 to 95%) with CD63 at all time points. This experiment was performed twice, with similar results. Similar results were also obtained with LAMP1 and LAMP2 and when MDM were used.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Quantitation Assay, Immunofluorescence, Staining, Infection, Incubation, Fluorescence, Microscopy

Journal:

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/IAI.72.6.3204-3217.2004

Figure Lengend Snippet: Immunoelectron microscopy demonstrates that CD63, but not cathepsin D, is present on F. tularensis phagosomes, whereas both markers are present on latex bead phagosomes in human MDM 4 h after infection. Human monocytes were isolated from peripheral blood, differentiated for 5 days in Teflon wells, plated on tissue culture plastic, incubated with the F. tularensis RCI and latex beads as described in the text, and fixed and prepared for cryoimmunoelectron microscopy. (A) Sections were stained with immunogold for CD63 (5-nm-diameter immunogold; arrowheads) and F. tularensis (15-nm-diameter immunogold; arrows). Both the F. tularensis phagosome and the latex bead phagosome stained positively for CD63. (B) Sections were stained for cathepsin D (5-nm-diameter immunogold particles; arrowheads) and F. tularensis (15-nm-diameter immunogold particles; arrows). Whereas the latex bead phagosomes had abundant staining for cathepsin D, the bacterial phagosomes had no staining for cathepsin D. Some F. tularensis antigen (15-nm-diameter gold) was also present in compartments outside of the phagosomes (larger, open arrows).

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Immuno-Electron Microscopy, Infection, Isolation, Incubation, Microscopy, Staining

Journal:

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/IAI.72.6.3204-3217.2004

Figure Lengend Snippet: Quantitation of immunogold staining of F. tularensis RCI phagosomes 4 h after infection of human MDM. Histograms demonstrate the distribution of immunogold staining for CD63 (top) and cathepsin D (bottom) in phagosomes fixed 4 h after coincubation with the F. tularensis RCI and latex beads. Whereas the majority of both F. tularensis phagosomes and latex bead phagosomes acquired abundant staining for CD63, only latex bead phagosomes showed abundant staining for cathepsin D. Control sections incubated with isotypic control mouse myeloma Igs had <0.25 gold particles per μm of membrane. The experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Quantitation Assay, Staining, Infection, Incubation

Journal:

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/IAI.72.6.3204-3217.2004

Figure Lengend Snippet: Killed, but not live, F. tularensis RCI bacteria enter acidified compartments that stain positive for DAMP in human MDM. Human MDM were fixed 3 h after infection with formalin-killed (A) or live (B) F. tularensis RCI, and acidified compartments were identified by DAMP immunogold staining (15-nm-diameter gold particles; arrows) and immunoelectron microscopy as described in the text. F. tularensis antigen was identified by immunostaining with 5-nm-diameter gold particles (arrowheads). Abundant staining for DAMP was associated with killed F. tularensis (A) but not live F. tularensis (B). This experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Staining, Infection, Immuno-Electron Microscopy, Immunostaining

Journal:

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/IAI.72.6.3204-3217.2004

Figure Lengend Snippet: Calculation of phagosomal pH by quantitative immunogold staining. Human MDM were infected with the live or killed F. tularensis RCI as described for Fig. ​Fig.6,6, and DAMP immunogold particles were enumerated in the bacterial compartments and in a reference compartment (the nucleus). The pH was calculated as described in Materials and Methods. Values shown are the means ± standard errors of at least 20 bacterial compartments from each of at least two electron microscopy grids. The experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Staining, Infection, Electron Microscopy

Journal:

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/IAI.72.6.3204-3217.2004

Figure Lengend Snippet: F. tularensis LVS and RCI are within vacuoles with clearly discernible membranes at early times after infection, but not at late times after infection. THP-1 cells were incubated for 90 min with the F. tularensis LVS (A to D) or RCI (E to H) and fixed immediately (A and E) or after an additional 3 h (B and F), 6 h (C and G), or 14 h (D and H). Monolayers were fixed with osmium tetroxide and glutaraldehyde, stained with uranyl acetate, embedded in Epon resin, thin sectioned, enhanced for contrast with uranyl acetate and lead citrate, and viewed by electron microscopy. Host cell membranes were well preserved in all sections. Bacteria are indicated by asterisks. Immediately after infection, the majority of F. tularensis LVS (A) and RCI (E) bacteria resided in compartments with easily discernible phagosomal membranes. Many bacterial phagosomal membranes had a thick fibrillar coat radiating approximately 30 nm from the cytoplasmic aspect of the membrane (A, B, E, and F; solid arrowheads). In other cases, the phagosomal membranes lacked these coats (e.g., white arrowheads in panel G). The coated membranes appeared to form buds (B and F [lower insert], open arrowheads), to pinch off and form vesicles (B and C, solid arrows), or to fragment (G, open arrows). By 14 h after infection, the majority of LVS (D) and RCI (H) bacteria (asterisks) lacked any identifiable phagosomal membranes. In all cases, the bacteria were separated from the host cell cytoplasm by electron lucent zones. Bars, 0.5 μm. This experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Infection, Incubation, Staining, Electron Microscopy

Journal:

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/IAI.72.6.3204-3217.2004

Figure Lengend Snippet: Quantitation of bacteria surrounded by phagosomal membranes. THP-1 cells were infected with the F. tularensis LVS or RCI and prepared for transmission electron microscopy as described in the text. Bacteria were scored as having a phagosomal membrane if at least 50% of their circumference was surrounded by a morphologically discrete membrane bilayer. Eighty to 90% of the bacteria had identifiable membrane bilayers when they were fixed immediately after a 90-min incubation with THP-1 cells (0 h); the percentage dropped to <20% by 14 h after infection. In the first 6 h after infection, 40 to 50% of the RCI phagosomes and 58 to 88% of the LVS phagosomes had densely staining fibrillar coats (data not shown). At 14 h, it was difficult to quantify the fraction of phagosomes with fibrillar coats because very few phagosomal membranes were visible. This experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Quantitation Assay, Infection, Transmission Assay, Electron Microscopy, Incubation, Staining