Joris van Dongen

135 Isolation of SVF by the FAT procedure 11. Incubate samples in phosphomolybdic-phosphotungstic acid for 12 min. 12. Wash samples in demiwater until color disappears. 13. Incubate samples in aniline blue for 7 min. 14. Wash samples in demiwater for 2 min. 15. Incubate samples in 1% acetic acid for 5 sec. 16. Wash samples in demiwater until color disappears. 17. Dry samples for 20 min. 18. Mount samples in Permount. OPTIONAL: Enzymatic solation of cellular SVF (cSVF) derived from tSVF 1. Follow step 1. till 12. of section 3.2. 2. Wash the isolated tSVF with PBS three times. 3. Add 0.1% collagenase A solution 1:1 with tSVF. 4. Stir the collagenase/tSVF mixture in a water bath at 37°C for 1.5h. 5. Centrifuge the sample at 600 xg at room temperature for 10 min. 6. Remove supernatant. 7. Collect cell pellet in PBS/1% BSA. 8. Filter the collagenase/tSVF mixture through filters. 9. Centrifuge the sample at 600 xg at room temperature for 10 min. 10. Repeat step 6. 7. and 8. 11. Remove supernatant. 12. Collect cell pellet in 30 ml of PBS/1% BSA. 13. Put 15 ml of lymphoprep in a 50 ml tube. 14. Gently add the 30 ml of PBS/1% BSAwith the sample. 15. Centrifuge the sample at 1000 xg at 4°C for 20 min. and put the brake on 0. 16. Remove the upper layer. 17. Take cells from the interphase carefully. 18. Add PBS/1% BSA to the cells.