Isolation and Propagation of Rat Peritoneal Mesothelial Cells

Antibody

IgG source

Dilution

Application

Company

α-SMA

Mouse

1:200

WB/IF

Sigma

Cytokeratin 18

Mouse

1:100

WB/IF

Sigma

Vimentin

Mouse

1:100

WB/IF

Sigma

Alexa 488

Goat

1:1000

Invitrogen

WB, western blotting; IF, immunofluorescence

Fixatives: methanol, 4 % PFA.

Permeabilizing solution: Triton X-100.

Blocking buffer: 1× PBS containing 3 % BSA (w/v) and 10 % goat serum (v/v).

Nuclei staining solution: Prepare a stock solution by dissolving 1 mg DAPI (4′, 6-diamidino-2-phenylindole) powder in 1 mL ddH₂O. Store at 4 °C in dark. To prepare working solution, dilute DAPI stock 1:1000 in ddH₂O and filter to remove undissolved material prior to use.

Anti-fade mounting medium.

Immunofluorescent or confocal microscope.

3 Methods

3.1 Preparation

3.1.1 Medium and Reagent

Preheat enriched medium and maintenance medium in 37 °C water bath.

Fill a 10-cm dish with 10-mL PBS, and place on ice before surgery.

3.1.2 Collagen Coating of Culture Dishes

Add 5 mL of coating solution (collagen solution) to each 25-cm² flask, and incubate for 30 min prior to starting.

3.2 Isolation of Mesothelial Cells from Rat Peritoneum

3.2.1 Isolation of Rat Peritoneal Mesothelial Cells by Intraperitoneal Injection

This procedure is performed with a modification of the technique reported by Shostak [12]. The experimental protocols were approved by the Animal Care and Use Committee of the Sun Yat-sen University.

Humanely kill rats with an overdose of anesthetic. When dead, quickly inject intraperitoneally with 10 mL/100 g bodyweight of 0.25 % trypsin in EDTA. Place the animal in a faceup position for 10 min, and then facedown 10 min.

To harvest RPMCs, the abdominal fluid of individual rats is collected in a 50-mL tube in a biohazard hood. Collect about 15–20 mL cell suspension for each rat.

Centrifuge cell suspension at 180 × g for 10 min at 4 °C.

Remove the supernatant, wash cell pellets with 25 mL D-Hanks’ balanced salt solution once to remove the trypsin, and repeat using the same centrifuge speed and time as above.

Resuspend the cell pellets in 25 mL maintenance medium (see Note 2 ).

Centrifugate again at 180 × g for another 10 min, suspend in 15-mL tube with 10 mL enriched medium, and gently pipette the mixture to resuspend cells.

Use a hemocytometer to count viable cells, based on trypan blue exclusion.

Aspirate the collagen solution off the 25-cm² culture flasks, and add 4 mL enriched medium to each 25-cm² flask.

Seed cells at 1–2 × 10⁶ with a final volume of 5 mL enriched medium in 25-cm² tissue culture flask. Incubate at 37 °C in a humidified 5 % CO₂ atmosphere.

10.

Refresh the medium when cells adhere to the surface of the flask (see Note 3 ).

3.2.2 Isolation of Rat Peritoneal Mesothelial Cells by Dissection of Peritoneum

Humanely kill rats with an overdose of anesthetic, and collect specimens of peritoneum (mainly mesenterium). Put peritoneal tissue in a 10-cm dish with 10 mL PBS on ice.

Wash tissue specimens extensively with PBS three times to remove contaminating red blood cells.

Dissect a 2–3-cm² piece of peritoneum with sharp ophthalmic scissors (see Note 4 ).

Digest the tissue with pre-warmed enzyme dissociation solution (0.1 % trypsin/0.02 % EDTA) for 30 min at 37 °C with continuous rotation.

Sieve the cell suspension with 70-μm strainer and collect the sieved solution (see Note 5 ).

Spin down the cell suspension by centrifugation at 180 × g for 10 min at 4 °C.

Wash the cell pellet twice in maintenance medium (use the same rpm and time as in the step above), and gently pipette the mixture up and down a few times.

Use a hemocytometer to count viable cells, based on trypan blue exclusion.

Seed cells at 1–2 × 10⁶ with a final volume of 5 mL enriched medium in 25-cm² tissue culture flask. Incubate at 37 °C in a humidified 5 % CO₂ atmosphere.

10.

Refresh the medium every 2–3 days.

These two methods have proven to be both efficient and reproducible for isolating rat peritoneal mesothelial cell s (see Note 6 ).

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