rabbit anti-ct momp antibody  (Virostat Inc)

Journal: Scientific Reports

Article Title: Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

doi: 10.1038/srep23148

Figure Lengend Snippet: ( A ) HeLa cells were infected with Ct L 2 and fixed at 24 hpi. The inclusion was labeled with anti- Ct MOMP antibody (green), ACSL-specific antibodies (red), and Hoechst for nuclear and bacterial DNA (blue) (see Methods). Representative images of z-stack projections from confocal microscopy are shown. White lines indicate localization of the ACSLs inside the inclusion in the three planes, x, y, and z. Scale bar, 5 μm; ( B ) HEp2 cells were infected with Ct L 2 for 24 h, and prepared for TEM. ACSLs were labeled with specific primary antibodies and secondary antibodies conjugated to 12 or 18 nm gold particles (see Methods). The Ct inclusion is shown in the upper panel and an inset at higher magnification in the lower panel. Arrows indicate the gold immunolabeling of respective ACSLs. Inc, inclusion; Cyt, cytoplasm. Scale bar, 500 nm; ( C ) HEp2 cells were infected with Ct L 2 for 24 h and labeled with an anti-ACSL3 antibody as above. A portion of the Ct inclusion is shown on the top panel (scale bar, 500 nm) and a higher magnification inset on the bottom panel. IB, intermediate body. Scale bar, 200 nm.

Article Snippet: Cells were stained with a rabbit anti -Ct MOMP antibody (Virostat) and imaged using confocal microscopy, as described previously.

Techniques: Infection, Labeling, Confocal Microscopy, Immunolabeling

Journal: Scientific Reports

Article Title: Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

doi: 10.1038/srep23148

Figure Lengend Snippet: ( A ) HeLa cells were treated for 16 h with the following ACSL activity inhibitors: 5 μM and 7.5 μM Triacsin C (TC); 200 μM and 300 μM 2-Fluoropalmitic acid (2-FPA), and 50 μM and 100 μM Rosiglitazone (RG). The cells were lysed, and ACSL activity was measured as fluorescent acyl-CoA recovered. Error bars indicate standard deviation for three independent experiments. The asterisks indicate statistically significant differences at ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by the two-tailed t-test; ( B ) HeLa cells were treated with the inhibitor TC at the indicated concentrations for 16 h and then infected with Ct L 2 , without removing the inhibitor from the media. At 24 hpi, the cells were fixed and prepared for confocal microscopy. Labeling was done with anti- Ct MOMP antibody (green) and with Hoechst to stain for nuclear and bacterial DNA (blue). Scale bar, 10 μm; ( C ) HeLa cells were treated with 7.5 μM TC for 16 h and infected with Ct L 2 for 24 h, without removing the inhibitor from the media. The cultures were used for reinfecting new HeLa cell monolayers and analyzed for infectivity and production of progeny. Values (mean ± standard error for three independent experiments) are shown as inclusion forming units (IFU)/mL. Asterisks denote statistically significant differences at ∗∗ p < 0.01, by the two-tailed t-test. The same experiment was carried out with the inhibitor 2-FPA ( D , E ) and RG ( F , G ) at noted concentrations; ( H ) HeLa cells treated with the inhibitors and infected with Ct as described previously were stained with anti- Ct MOMP antibody and imaged using confocal microscopy. The images were analyzed using Imaris X64 software to identify Regions Of Interest (ROI) coinciding with the inclusions. The software was used to calculate the mean inclusion area (in μm 2 ) based on the ROIs. Values (mean ± standard error for three independent experiments) are shown. The asterisks indicate statistically significant differences between each condition compared to the control by the two-tailed t-test ( ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001).

Article Snippet: Cells were stained with a rabbit anti -Ct MOMP antibody (Virostat) and imaged using confocal microscopy, as described previously.

Techniques: Activity Assay, Standard Deviation, Two Tailed Test, Infection, Confocal Microscopy, Labeling, Staining, Software, Control

Journal: Scientific Reports

Article Title: Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

doi: 10.1038/srep23148

Figure Lengend Snippet: DGAT2−/− MEF cells were treated with the inhibitor T863 or left untreated in the presence of 100 μM OA to stimulate the formation of LDs. After 16 h of incubation, cells were left uninfected ( A ) or infected with Ct L 2 ( B ) without removing the inhibitor from the media. After 24 hpi, cells were fixed and labeled with anti- Ct MOMP antibody (red), BODIPY 493/503 for neutral lipids (green), and Hoechst for nuclear and bacterial DNA (blue). Scale bar, 10 μm.

Article Snippet: Cells were stained with a rabbit anti -Ct MOMP antibody (Virostat) and imaged using confocal microscopy, as described previously.

Techniques: Incubation, Infection, Labeling

Journal: Scientific Reports

Article Title: Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

doi: 10.1038/srep23148

Figure Lengend Snippet: Control and T863 treated DGAT2−/− MEF cells were infected with Ct L 2 without removing the inhibitor from the media. After 24 h, cells were fixed and labeled with anti- Ct MOMP antibody (green), anti-ACSL3 ( A ) or anti-ACSL4 ( B ) antibodies (red), and Hoechst to stain nuclear and bacterial DNA (blue). Representative images of z-stack projections from confocal microscopy are shown. White lines indicate localization of ACSLs within the Ct inclusion in the three planes, x, y, and z. Scale bar, 5 μm.

Article Snippet: Cells were stained with a rabbit anti -Ct MOMP antibody (Virostat) and imaged using confocal microscopy, as described previously.

Techniques: Control, Infection, Labeling, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

doi: 10.1038/srep23148

Figure Lengend Snippet: TC ( A ), 2-FPA and RG ( B ) were added to control and T863 treated DGAT2−/− MEF cells at two different concentrations (TC: 5 μM and 7.5 μM; 2-FPA: 200 μM and 300 μM; RG: 50 μM and 100 μM) for 16 h. The cells were then infected with Ct L 2 without removing the inhibitor from the media. After 24 hpi, samples were prepared for WB analysis. Membranes were probed with anti- Ct HSP60 antibody and anti-human GAPDH antibody as a loading control. ( C ) DGAT2−/− MEF cells were treated with the inhibitor T863 (lower panels) or left untreated (upper panels). After 8 h of incubation, the following ACSL activity inhibitors were added: 100 μM RG, 300 μM 2-FPA, and 7.5 μM TC. After 16 h of incubation, cells were infected with Ct L 2 , without removing the inhibitors from the media. After 24 hpi, cells were fixed and visualized by confocal microscopy. Cells were stained with anti- Ct MOMP antibody (red), BODIPY 493/503 for neutral lipids (green), and Hoechst for nuclear and bacterial DNA (blue). Scale bar, 10 μm.

Article Snippet: Cells were stained with a rabbit anti -Ct MOMP antibody (Virostat) and imaged using confocal microscopy, as described previously.

Techniques: Control, Infection, Incubation, Activity Assay, Confocal Microscopy, Staining

Journal: Scientific Reports

Article Title: Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

doi: 10.1038/srep23148

Figure Lengend Snippet: ( A ) HeLa cells were infected with Ct L 2 and were either treated with Brefeldin A (BFA) from 2–4 hpi (right) or left untreated (left). After 24 hpi, cells were fixed and prepared for confocal microscopy. The inclusion was labeled with anti- Ct MOMP antibody (green), anti-human golgin 84 antibody (red), and Hoechst for nuclear and bacterial DNA (blue). Scale bar, 10 μm; ( B ) HeLa cells were infected with Ct L 2 and treated with Brefeldin A (BFA) from 2–4 hpi. After 24 hpi, cells were fixed and prepared for confocal microscopy. The inclusion was labeled with anti- Ct MOMP antibody (green), anti-human ACSL-specific antibodies (red), and Hoechst for nuclear and bacterial DNA (blue). Representative images of z-stack projections from confocal microscopy are shown. White lines indicate localization of the ACSLs inside the inclusion in the three planes, x, y, and z. Scale bar, 5 μm.

Article Snippet: Cells were stained with a rabbit anti -Ct MOMP antibody (Virostat) and imaged using confocal microscopy, as described previously.

Techniques: Infection, Confocal Microscopy, Labeling