Difference between revisions of "Isolating murine prostate smooth muscle cells"
(New page: ==Mouse prostate SMC isolation== =Reagents= 1. Collagenase Type II (Worthington Biochemical LS004174) 2. Elastase (Worthington Biochemical LS002279) 3. Soybean Trypsin Inhibitor (Worthin...)
Revision as of 06:19, 19 March 2012
Mouse prostate SMC isolation
1. Collagenase Type II (Worthington Biochemical LS004174) 2. Elastase (Worthington Biochemical LS002279) 3. Soybean Trypsin Inhibitor (Worthington Biochemical LS003570) 4. HBSS (with calcium and magnesium) 5. DMEM/F12 medium (Gibco 11320) 6. Penicillin/Streptomycin 7. Fetal Bovine Serum
Enzyme Solution [make fresh on day of isolation]
1. 10 mg collagenase to a final concentration of 1 mg/ml 2.10 mg soybean trypsin inhibitor to a final concentration of 1 mg/ml 3. 10 ml HBSS 4. ca. 70 μL elastase to a final concentration of ca. 0.7-0-75 units/ml 5. 100 μL pen/strep to a final concentration of 1%
Medium 1. 1X DMEM/F12: 400 ml 2. Penicillin/Streptomycin: 5 ml (1% final) 3. Fetal Bovine Serum (bought heat inactivated): 100 ml (20 % final) 4.Filter
1. Use of younger male mice (6-8 weeks) is preferred to older males. 2. Put enzyme solution (2.5 ml) in a well of a 6well plate, next to it add 2.5 ml medium (containing 20% FBS), next to it another well with 2.5 ml medium, Equlibrate in incubator (37°C, 5%CO2) 3. Prepare 5 ml HBSS in 15 ml Falcon tube, put on ice. Prepare HBSS in 60 mm dish for tissue preparation
Isolation of prostate lobes
4. Kill mouse by cervical dislocation 5. Spray with 70%ethanol, open skin over abdomen with sterile scissors, open muscle tissue to face abdominal organs (best place to cut is lower abdominal region) 6. Take out the bladder by holding onto it with forceps; pull organs attached to bladder, including urethra, prostate, seminal vesicles (SV) out. Cut urethra and connective tissue below to pull out whole periurethral region. Put into 60 mm dish with HBSS. 7. Using a stereo microscope, dissect prostate lobes. First, remove all non-prostate tissue and connective tissue: remove ductus deferens, ampullary gland (if present), and all fat tissue (resembles Styrofoam in appearance). Keep prostate tissue (transparent glandular tissue). Prostate lobes can be found: the anterior prostate is the biggest lobe and lines the inner curvature of the SV. The dorsal prostate is attached to both the initial segment of the outer curvature of the SV and the urethra. The lateral and ventral prostates are located above the dorsal lobe, only connected to the urethra. Cut these lobes at the insertion points to remove them. Put all lobes in HBSS in flacon on ice, and then move to cell culture hood.
Initial digestion and tissue preparation
8. Take prostate lobes from Falcon tube and put into pre-equilibrated enzyme solution. Incubate for 8-10’ at 37°C in the incubator. 9. Transfer tissue from enzyme solution to the first well filled with medium (DMEM/F12+20%FBS, 1%P/S). Using observation under a stereo microscope, spread the single ducts within a prostate lobe; i.e., hold down the lobe with one forceps and, using a thin forceps, streak along the in between two ducts. This will remove fibrous material (collagen + fibroblasts) from the tissue. 10. Transfer the spreaded ductal tissue to the second medium-filled well to wash it.
Final digestion and tissue trituration
11. Transfer the tissue to a new dish (e.g., 1 well of a 6well plate, 2.5 ml enzyme solution) with enzyme solution. Incubate at 37°C, 5%CO2 in incubator for about an hour. 12. In the meantime, prepare fire-polished Pasteur pipettes: Round the edges of the Pasteur pipette outlet by shortly heating it over a flame, quickly turning it. 13. Triturate the cells by pipetting up and down with fire-polished Pasteur pipette (15-20x). 14. Transfer enzyme solution + triturated tissue together with 5 ml of gowth medium into 15ml conical tube (Falcon). Spin 5’ 500g. 15. Wash by aspirating supernatant, but leave about 1ml in tube in order to avoid aspirating the tissue / cells. Add 10 ml medium. Repeat 2x. 16. Suspend cells / tissue in ca. 20 ml medium per prostate, plate on either 24 wells of a 48well plate, 6 wells of a 12 well plate, 3 wells of a 6 well plate. Leave undisturbed for 5-7 days. Then change medium, split cells when very dense.