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E7889

Manufactured by Merck Group
Sourced in United States, Sweden

The E7889 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of this product is to provide a controlled environment for various experiments and processes. Further details on the intended use or specifications of this product are not available.

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2 protocols using e7889

1

Immunofluorescent Characterization of VSMCs

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VSMC cultures were trypsinized at cell density of 200,000 cells/5 mL in T25 flasks and then fixed with 4% paraformaldehyde during 10 min at room temperature. VSMCs were permeabilized with 0.5% saponin (S7900, Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 5 min and blocked with 1% BSA (A6003, Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Primary antibodies used were anti-alpha smooth muscle actin at 1/100 (ab66133, Abcam, Cambridge, UK), transgelin/SM22 monoclonal antibody at 1/100 (60213-1-Ig, ProteinTech, Manchester, UK) and anti-CD31 (also called platelet and endothelial cell adhesion molecule 1, PECAM1) [EPR3094] antibody at 1/50 (ab76533, Abcam, Cambridge, UK) during 1 h in shaking at room temperature. VSMCs were washed with 5 mM EDTA (E7889, Sigma-Aldrich, St. Louis, MO, USA) and 0.5% BSA in PBS and were incubated with secondary antibodies Alexa Fluor® 488 goat anti-mouse IgG (H + L) (A-11001, Invitrogen, Thermofisher, Carlsbad, CA, USA) and Alex Fluor® 647 goat anti-rabbit IgG (H + L) (A-21244, Invitrogen, Thermofisher, Carlsbad, CA, USA) at 1/500 for 30 min in shaking and darkness. After washing, cells were resuspended in 5 mM EDTA and 0.5% BSA in PBS.
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2

Isolation of Immune Cells from Mice

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Mice were deeply anesthetized by injecting 100 μg pentobarbital intraperitoneally. Blood was collected from the right ventricle prior to perfusion. Briefly, 200 μL blood were collected into tubes containing ethylenediaminetetraacetic acid (EDTA, E7889, Sigma, Sweden), lysed in ACK buffer (A1049201, Gibco) and then centrifuged. The pellet was resuspended in cold PBS and used for staining. Spleens were dissected and cell suspensions prepared by mechanical dissociation in ice-cold PBS by passing through 40 μm cell strainers (734-0002, VWR; Stockholm, Sweden). Mice were perfused through the left cardiac ventricle using ice-cold PBS. Whole brain and spinal cord were removed and minced with a surgical disposable scalpel (Medicarrier AB, Sweden), followed by enzymatic digestion using Collagenase (11088866001, Roche, Sweden) and DNAse (000000010104159001, Roche, Sweden). Myelin was removed using 38% Percoll (P1644-1L, Sigma, Sweden).
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