Mouse Islet Culture and si RNA Protocol
(Day 1) Typically we obtain 1200 mouse islets from 12 Cd1 mice. The isolation is done by the UVA Islet Isolation Core. Islets are delivered to us in RPMI + P/S + 10% FBS with 25 mm Glucose (which is prepared by us and given to the islet core lab).
Media Recipe: RPMI (Gibco #11875-093), which contains 11.1 mM glucose. We add 5 ml Penn Strep, 50 ml FBS and 6.9 ml of sterile 1M glucose to make the final glucose concentration 25 mm
Islets are plated in 10 cm Petri dishes in RPMI media (above) and allowed to recover from isolation.
(Day 2): Islets are pooled using siliconized pasteur pipets in a sterile 15 ml blue cap tube, centrifuged 500 RPM for 1 min. Media is aspirated and pellets are gently washed in 5 ml PBS, centrifuged 1 min at 500 RPM.
Islet pellets are resuspended in serum free RPMI plus P/S such that 800 mL contains approx 150 islets and islets are plated in 6 well tissue culture dishes and incubated for 2 hrs.
Remember to plate 150 islets each for RNA and protein for the time=0. Harvest at the time of transfection.
Transfection: Desired amount of adenovirus is added to each well and incubated for 2 Hrs and then 1.2 mL serum containing media is added to each well and islets are incubated O/N. The controls with no si-RNA should also be treated the same way. Plate 150 islets in 800 ml SFM and add 1.2 ml medium later just like the transfected islets. Do not bring the control islets to the Ad-viral incubator-keep separately.
(Day 3): After 24 hrs (from the time of infection), add 1 ml of serum containing media to the islets to make the total volume 3 ml. Incubate for an additional 2 days. (Total time incubated with virus is 72 hrs). (In this protocol, we don’t remove the virus from the culture medium -we just dilute it).
(Day 5): Harvest islets in 350 ml RLT buffer/well for RNA and protein.
After 72hrs, collect the medium from the wells into a 15-ml tube. Wash the wells with 1 ml sterile PBS twice and pool to the medium. (15-ml tube now contains 3ml medium+2ml PBS). Add 350 ml RLT buffer (RNA) OR 100 ml of 1X SDS sample buffer with out blue dye (protein) to the wells and keep. (50ml of 2XSDS buffer + 50 ml of PBS to make 1X SDS buffer, avoid blue to help measurement of protein).
Now, spin the 15-ml tube for 5 min at 1000 rpm. Remove the supernatant and resuspend the pellet in 1 ml PBS and transfer to a 1.5 ml eppendorf tube. Spin again for 5 min at 1000 rpm. Remove the supernatant and keep the pellet on ice.
Scrape the wells (RLT or SDS buffer) with a scraper and transfer the lysates to the eppendorf tubes (which contain the pellets) using a syringe. Now mix the pellet with the lysates you collect from the wells. Pass 6-7 times through the needle. Freeze the sample at –80 for later use.
Confirm the knock down of Pdx1 by Western Blot and real-time RT-PCR
