Human alpha thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Lipopolysaccharide (LPS) from E. coli, diethylaminoethyl (DEAE)-dextran, and 4-phenylbutyrate (4-PBA) were purchased from Sigma-Aldrich Chemical (St. Louis, MO). Polyclonal antibodies to VCAM-1, ICAM-1, IκBα, RelA/p65, and β-actin were from Santa Cruz Biotechnology (CA). Antibodies to VE-cadherin were purchased from Abcam (Cambridge, MA) and BD Biosciences (San Jose, CA). BiP/GRP78 polyclonal antibody was obtained from Cell Signaling Technology (Beverly, MA). Expression vector encoding Wild type BiP/GRP78 and dominant negative BiP/GRP78 were from Addgene. SubAB, SubA and the non-active derivative SubAA272B were purified as previously described28 (link),29 (link),46 (link). All other materials were from VWR Scientific Products Corporation (Gaithersburg, MD) and Fisher Scientific (Pittsburgh, PA).
Diethylaminoethyl deae dextran
Manufactured by Merck Group
Sourced in United States, Sao Tome and Principe
Diethylaminoethyl (DEAE)-dextran is a laboratory reagent used as a transfection agent. It facilitates the introduction of nucleic acids, such as DNA or RNA, into cells. DEAE-dextran functions by forming a complex with the nucleic acid, which can then be taken up by the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Human α thrombin, by Enzyme Research (9 mentions)
Plasmid maxi kit, by Qiagen (3 mentions)
Human alpha thrombin, by Enzyme Research (3 mentions)
Antibodies to ve cadherin, by Abcam (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Anti phospho ser3 cofilin1 antibody 3311s, by Cell Signaling Technology (2 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (2 mentions)
Lipopolysaccharide lps from e coli, by Merck Group (1 mentions)
4 phenylbutyrate 4 pba, by Merck Group (1 mentions)
Rela p65, by Santa Cruz Biotechnology (1 mentions)
Icam 1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Agarose, by Merck Group (1 mentions)
Agarose, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by Merck Group (1 mentions)
9 protocols using diethylaminoethyl deae dextran
1
Modulation of Endothelial Cell Stress Pathways
2
Measuring Yeast Cell Wall Porosity
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The relative porosity to polycations was used to calculate the wall porosity, as described previously.28 (link)
S. cerevisiae cells were grown in SD-Gal broth until they reach the midlog phase, washed twice with PBS, and cell concentration adjusted at 1×108 cells/mL. Aliquots containing 1 mL were centrifuged, the supernatant discarded, and cell pellets were suspended in either 10 mM Tris-HCl, pH 7.4 (buffer A), buffer A plus 30 µg/mL poly-l-lysine (MW 30–70 kDa; Sigma-Aldrich Co.) or buffer A plus 30 µg/mL diethylaminoethyl (DEAE)–dextran (MW 500 kDa; Sigma-Aldrich Co.). Cells were incubated for 30 minutes at 28°C with shaking at 200 rpm, centrifuged, and the supernatants were saved, centrifuged again, and the absorbance at 260 nm was measured. The relative cell wall porosity to DEAE–dextran was calculated as reported previously.28 (link)
S. cerevisiae cells were grown in SD-Gal broth until they reach the midlog phase, washed twice with PBS, and cell concentration adjusted at 1×108 cells/mL. Aliquots containing 1 mL were centrifuged, the supernatant discarded, and cell pellets were suspended in either 10 mM Tris-HCl, pH 7.4 (buffer A), buffer A plus 30 µg/mL poly-l-lysine (MW 30–70 kDa; Sigma-Aldrich Co.) or buffer A plus 30 µg/mL diethylaminoethyl (DEAE)–dextran (MW 500 kDa; Sigma-Aldrich Co.). Cells were incubated for 30 minutes at 28°C with shaking at 200 rpm, centrifuged, and the supernatants were saved, centrifuged again, and the absorbance at 260 nm was measured. The relative cell wall porosity to DEAE–dextran was calculated as reported previously.28 (link)
3
Thrombin-Induced Signaling Pathway Analysis
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Human α-thrombin was purchased from Enzyme Research Laboratories (South Bend, IN). Anti-ATG7, anti-phospho-(Ser536)-RelA/p65 (3033S), anti-phospho-(Ser3)-Cofilin1 antibody (3311S), and anti-Cofilin1 (clone D3F9, 5175S) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to RelA/p65 (SC-8008), IκBα (SC-371), and β-actin (SC-47778) were from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-TBP antibody was purchased from Abcam (AB33168, Cambridge, MA). Plasmid maxi kit was from QIAGEN Inc. (Valencia, CA) and diethylaminoethyl (DEAE)-dextran was obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). All other materials were obtained from Thermo Fisher Scientific (Waltham, MA).
4
DEV Virus Plaque Titration in CEF Cells
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CEF cells were washed once with phosphate-buffered saline (PBS) before being infection with the DEV virus. The inoculum was removed at 2 h post-infection and replenished with either fresh medium or 2% Minimum Essential Medium (MEM)-agarose overlay. The MEM-agarose overlay medium contains MEM (Sigma, St Louis, MO, USA), 2% agarose (Thermo Fisher Scientific, Waltham, MA, USA), 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM l -glutamine (Sigma), 0.3% bovine serum albumin (BSA) (Sigma), 15 mM 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) (Sigma), 0.22% sodium bicarbonate (Sigma) and 0.01% Diethylaminoethyl (DEAE)-Dextran (Sigma). For the virus plaque titration, infected cells were incubated at 37 °C under a 5% CO2 atmosphere for 5 days before being fixed with 1% crystal violet (Sigma) in 20% ethanol for plaque counting and plaque size measurement.
5
Assay for HIV-1 Entry Inhibitors
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HEK-293T cells were purchased from ATCC (Manassas, VA). U87-CD4-CXCR4 cells, U87-CD4-CCR5 cells, TZM-bl cells, MT-2 cells, CHO-WT cells, T20, AMD3100, the pNL4-3E-R-Luc plasmid, HIV-1, and vesicular stomatitis virus-G (VSV-G) Env-encoding plasmids were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. The peptides N36 and C34 were synthesized by a standard solid-phase Fmoc method by GL Biochem of China and were purified by HPLC. ADS-J1 was purchased from ComGenex (Budapest, Hungary). Mouse mAb NC-1 specific for the gp41 6-HB was prepared and characterized as previously described (Jiang et al., 1998 (link)). Horseradish peroxidase (HRP)-labeled goat anti-mouse-IgG and goat anti-rabbit-IgG were purchased from Invitrogen (San Francisco, CA). Polyethyleneimine (PEI), biotin-labeled goat anti-mouse IgG, 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino) carbonyl-2H-tetrazolium hydroxide [XTT], diethylaminoethyl (DEAE)-dextran and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO). Streptavidin-labeled horseradish peroxidase (SA-HRP) was purchased from Zymed (South San Francisco, CA).
6
Estimating Cell Wall Porosity
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The relative cell wall porosity to polycations was estimated as described previously.43 (link) Cells were grown until reaching the exponential phase, washed twice with PBS, and cell concentration was adjusted at 1×108 cells mL−1. Aliquots containing 1 mL were centrifuged, the pellet was saved and resuspended in either 10 mM Tris-HCl, pH 7.4 (buffer A), buffer A plus 30 µg·mL−1 poly-L-lysine (Mw 30–70 kDa, Sigma-Aldrich Co.), or buffer A plus 30 µg·mL−1 diethylaminoethyl (DEAE)-dextran (Mw 500 kDa, Sigma-Aldrich Co.). Cells were incubated for 30 minutes at 30°C with constant shaking (200 rpm), centrifuged, and the supernatants were saved, centrifuged again, and used to measure the absorbance at 260 nm. The relative cell wall porosity to DEAE-dextran was quantified as reported elsewhere.43 (link)
7
Antibody Characterization for Cell Signaling
8
Endothelial Cell Signaling Pathway
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Human α-thrombin was purchased from Enzyme Research Laboratories. Antibodies for VE-cadherin (#SC-9989 & #SC-6458); were from Santa Cruz Biotechnology. Syk (#13198) antibody was from Cell signaling Technology. Phospho-VE-cadherin (Tyr658) (#44-1144G) antibody was from Thermo Fisher. ICAM-1 (#AF796) and VCAM-1 (#AF643) antibodies were purchased from R&D Systems. Phospho-VE-cadherin (Tyr685) (#AB119785) antibody was purchased from Abcam. The Plasmid maxi kit was from QIAGEN Inc and diethylaminoethyl (DEAE)-dextran was obtained from Sigma. All other materials were obtained from Thermo Fisher Scientific.
9
Viral Plaque Assay and Purification
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10-fold serial dilutions of infectious samples were prepared in Opti-MEM 0.3% BSA and added to PBS-washed confluent Vero E6 cells, incubated for 2 h at 37°C with 5% CO2 before inoculum was removed, and cells overlaid with DMEM containing 0.1% BSA, 1.5% Avicel (FMC BioPolymer), 0.5 mg/ml L-glutamine (Roth), 20 mM Hepes, 20 U/ml penicillin, and 20 µg/ml streptomycin and incubated for 72 h at 37°C with 5% CO2. After removal of Avicel medium, cells were fixed using 10% formalin solution and stained with 1% crystal violet in H2O containing 20% ethanol, each step for at least 25 min. Plaques were counted and used to calculate virus titers defined as PFU per ml.
To plaque-purify clonal isolates, cells infected with serial dilutions were overlaid with DMEM containing 2% FCS, 0.6% agar (Oxoid), 0.01% diethylaminoethyl (DEAE)-dextran (Sigma-Aldrich), and 0.1% NaHCO3 (Merck), and plaques picked 48–72 h p.i. using a thin filter-tip containing 2 µl PBS.
To plaque-purify clonal isolates, cells infected with serial dilutions were overlaid with DMEM containing 2% FCS, 0.6% agar (Oxoid), 0.01% diethylaminoethyl (DEAE)-dextran (Sigma-Aldrich), and 0.1% NaHCO3 (Merck), and plaques picked 48–72 h p.i. using a thin filter-tip containing 2 µl PBS.
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