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Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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Reagents

Saline solution G 10x: dissolve in 60 ml dH2O, 8 g NaCl, 0.4 g KCl, 0.016 g CaCl2•2H2O, 0.15 g KH2PO4, 0.154 g MgSO4•7H2O, 0.192 g Na2HPO4•2H2O, 1.1 g D-glucose, and 0.0012 g phenol red, waiting to each addition complete dissolution, and finally to bring to 100 ml with dH2O.

Transport medium at pH 7.2 at 300 ± 10 mOsm: in 100 ml graduate beacker dispense 10 ml of saline solution G 10x, and then fill up to 50 ml with dH2O, dissolve 0.05 g thimerosal, 0.1 g sodium bicarbonate, 0.5 g N-acetylcysteine, 0.1 g BSA and bring to 100 ml with dH2O. Store at 4 °C.

Percoll gradient: dissolve 0.1 g of sodium bicarbonate in 10 ml dH2O and the add 0.96 g of minimum essential medium (MEM). In other 30 ml of dH2O, dissolve 1 g of BSA (heat shocked and fatty-acid free) that is mixed with bicarbonate/MEM solution. At this solution add 57.4 ml of Percoll and fill up to 100 ml with dH2O. The resulting solution is frozen at below -20 °C for 48 hours. The Percoll solution is thawed at 4 °C for 48 hours before use.

All the solutions were usable for 2 - 3 weeks.

Procedure

Suspend 0.5 - 1.0 g fecal sample in 15 ml of transport medium, mixing thoroughly to ensure maximal preservation. Sample collected can be transported at room temperature (< 30 °C, for greater temperature cold pack is added), but must be processed within 7 days of collection to minimize loss of cell viability. If the samples cannot be processed immediately, store them at 4 °C.

Before beginning separation process, vortex the collection tube to suspend the sample. Filter the sample on double gauze veil or through a 330 µm plastic filter, and then filter the sample through 40 µm filter dividing 4 ml of filtered into 15 ml centrifuge tube, and add 6 ml of transport medium and mix by vortexing. Using a syringe 19 G Luer needle assembled, loaded with 4 ml pre-warmed to room temperature Percol gradient, dispense, slowly, in the bottom tube carefully underlay the sample avoiding mixing solution. Centrifuge the sample tubes at 200 g for 10 minutes at room temperature in a swinging bucket centrifuge with the brakes off. After centrifugation, remove supernatant, up to 1 cm above the interphase with Percoll gradient. Carefully remove the interphase including 1 ml of the supernatant from above the interphase and transfer in other tubes. Wash cell fractions by adding up to 15 ml, total volume, of cold PBS 0.1 M at pH 7.2 to each of the tubes. Re-suspend by either inverting the tubes several times or gently vortexing. Spin the cells at 900 g at 4 °C for 10 min. Carefully aspirate the supernatant and repeat wash twice, using 15 ml aliquots of PBS. Finally, re-suspend the pellet in 1 ml cold PBS and place the cell suspension on ice. To disaggregate clumped cells, pass cell suspension through a 20 G needle 10 times while the tubes are on ice. Mix the cells by tapping the tube gently to make the cells suspension homogenous. Count the cells in hemocytometer and evaluate viability with test esclusion trypan blue.