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Isolation of lymphocytes from the lamina propria (LPLs)

 

 

A. Materials

 

            * Serum Free Media: IMDM + 1X antibiotic-antimycotic (#15240062 gibco)

            * Media containing 10% FBS: IMDM + 10% FBS + 1X antibiotic-antimycotic

            * DNase from Sigma

* Liberase TL from Roche 

 

For 1-2 mice (2 mice can be pooled for this protcol):

 

* 50 ml tube.

* sterile stainless steel tissue sieve.

* 70 µm cell strainers

 

B. Procedure

 

            *Isolation of the large/small intestine and the Peyer’s patches.

            1. Euthanize the mice by cervical dislocation and perform midline incision and retract the skin.

2. Cut the intestine at the junction with the pyloric valve and slowly draw it out of the peritoneal cavity. Then cut the intestine at the junction with the cecum. While drawing it out, remove the fat with your fingers. This can also be accomplished using tweezers. Place the intestine in a Petri Dish containing ice cold PBS (should be on ice, too). This step is critical to obtaining a large and vibrant population of cells from this tissue!

            3. Remove the residual connective tissue and the fat from the intestine and flush out fecal matter using 10ml syringe with 200ul tip.

            4. Cut the small intestine longitudinally with fine scissors. Remove the feces from the gut by gently running fingers in parallel to the tissue. Then, wash the open small intestine several times in cold PBS, being certain to remove any impacted feces.

            5. Cut the open small intestine into 1-2 cm pieces and place the pieces in a 50 ml tube containing cold PBS.

            6. Vortex for 30 sec. and Tap sterile stainless steel tissue sieve. Repeat one more.

            7. Transfer new 50 ml tube containing 5mM EDTA in PBS

8. Incubate for 30 min at room temperature in a shaker.         

9. To begin to separate the intestinal epithelial and lamina propria (LP) layer: strain the content of the flask throught a sterile fine-meshed kitchen strainer into a 500 ml beaker held on ice. Tap strainer on beaker several times after straining and then transfer the pieces of intestine to a 50 ml conical tube containing cold PBS. Shake the tube vigorously for 30 seconds and then strain the content of the tube through the same kitchen strainer into the same beaker (containing the 20 ml collected previously).

10. Repeat the tapping/shaking/straining 2-3 more times.

11. Mince well the pieces of intestine by scissors in 1.5 ml tube.

 

 

            *Isolation of lamina propria lymphocytes.

 

            12. Put the intestine pieces recovered from step 11 into a sterile 50 ml tube and add 4 ml of serum free media/intestine + 1X liberase + 0.05% DNase (no serum!). Shake for 45 min at 37°C.

            13. Mash the intestinal pieces through a 70mm cell strainer placed atop a 50ml conical on ice.

            14. Centrifuge the cell suspension for 5 min at 300 G, 4°C. Resuspend in 1ml of serum free media and cell count.