Mouse Endothelial Cell Isolation Protocol

Disclaimer

Please note that this procedure is intended as a guideline only, and Creative Biolabs cannot guarantee specific results for the mouse endothelial cell isolation process.

Mouse endothelial cells are commonly used in biological research, and Creative Biolabs shares the use of the PECAM1 antibody in immunoprecipitation to isolate these cells from mouse organs. We also provide related products, such as Human PECAM1 membrane protein expressed in multiple expression systems. In terms of the novel Magic™ membrane protein production services, Creative Biolabs can produce membrane proteins in various formats to meet our clients' requirements. Comprehensive Immunogen Design Services are available for you.

Below are the general procedures for isolating mouse endothelial cells.

Procedures

Day 1: Preparation of magnetic beads for immunoprecipitation

The night before the harvest, prepare the following under a hood using the sterile technique:

Prepare Commercially available magnetic cell isolation beads: use 50 µL of magnetic beads for each harvested mouse.
Suspend the beads in 0.1% BSA (diluted with 1x PBS and filtered under a vacuum sterile hood), place them on a small magnetic separator, aspirate the supernatant, and wash the beads in a 1.7-mL eppendorf tube. Repeat this step twice.
Suspend the beads in 0.1% BSA (2 mL per 50 µL of beads) and load them into round-bottom 15 mL polystyrene tubes.
Add anti-mouse PECAM-1. Incubate each 50 µL containing 5 µL overnight at 4ºC on a rocker.

Day 2: Preparation of the mouses and organs for mouse endothelial cells isolation.

Euthanize the mouse by using CO₂.
Submerge the mouse in 70% ethanol.
Remove the heart, lungs, liver, spleen, and brain as aseptically as possible by using autoclaved instruments. Store the organs on ice in DMEM high glucose supplemented with 5 mL of penicillin/streptomycin.
Prepare collagenase type 1.
Dilute 2 mg of collagenase per mL of 1% BSA.
Add 1 µL of 1 M CaCl₂ and 1 µL of 1 M MgCl₂ per mL.
Use 25 mL of collagenase for each mouse sample. Collagenase must be sterile vacuum filtered in a hood prior to use.
Coat P100 dishes with gelatin and allow them to stand in the incubator for at least 30 min. The plates must be completely dry before applying cells.
The following steps should be performed with sterile equipment.
Transfer the organs to a petri dish and clean off any fat or excess tissue.
Use 2 sterile razors to mince the organs. Take care that this process does not take more than 1 minute.
Transfer the minced organs to a 50 mL tube containing 25 mL of collagenase and incubate in a 37ºC water bath for 1 h, shaking the mixture occasionally.
Fix the cannula to a 60 mL syringe and titrate the sample three times.
Transfer the mixture through a 70 µm disposable cell filter into a new 50 mL tube.
Centrifuge the 50 mL tube at 1,300 rpm for 5 minutes at 4ºC.
Aspirate the supernatant without destroying the cell pellet.
Resuspend the pellet in 25 mL of 0.1% BSA and centrifuge again at 1300 rpm for 5 minutes at 4ºC.
Aspirate the supernatant and resuspend the pellet in 1 mL of 0.1% BSA.
Place the polystyrene tube on a large separator to clean the PECAM-incubated beads, aspirate the supernatant, and re-inject 10-15 mL of 0.1% BSA. Repeat this step two more times.

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