DMEM (Cellgro catalog # 10-013-CV; 4.5g/L glucose) supplemented with 10 % heat inactivated FCS. I prefer to avoid any antibiotics in my cultures, but if necessary can be supplemented to this media.

Split cells 1 à 3

Cells can be used for step-down when they are ~ 80-90% confluent. If left unattended they tend to form some clusters on top of the monolayer of cells.

Step down

Differentiation of PANC1 cells

Generally I prefer to take 80-90% confluent flasks (T-80) for step-down protocol. The passage number obtained from ATCC was passage 57 and this will be referred to herein as passage 0. The cells that were first obtained from these are passage 1 and so on. In my experience, passages 3-7 have been the best candidates in achieving decent aggregation / differentiation of these precursor PANC-1 cells. Later passages have several issues in aggregation, wherein I did see that not all the cells at later passages contributed to aggregate formation; some single cells also remained attached as typical epithelial cells even in the serum-free medium (SFM).

Media composition:

Day 0 (day of step-down):

DMEM / F12: Mixed 1:1

DMEM was obtained from Gibco (Catalog # 11885-084)

F-12 medium was from Cellgro (Catalog # 10-080-CV)

BSA (ICN Biomedicals inc. Catalog # 152401, lot 3506F): 1%

Sodium selenite (Sigma S-5261; FW 172.9): 0.0067 mg/l

Transferrin (Gibco catalog # 13008-016; 4mg/ml stock): 5.5mg/l

IGF-1 (Calbiochem catalog # 407240; 10µg/ml stock): 10ng/ml

Day 4

Day 0 media

Taurine (Sigma catalog # T-8691; FW 125.1): 0.3mM

Day 10

Day 0 media* (everything else is same except BSA increased to 1.5%)

Taurine (Sigma catalog # T-8691; FW 125.1): 3.0mM

GLP-1 (Stock 100µM): 100 nM

Nicotinamide: 1mM

NEAA: 100 µM

Procedure:

Take passage 3-7 PANC-1 cells à remove SCM and add ~ 8ml Trypsin

Dilute the trypsin digest with SFM (day 0) at the end of the digest and spin cells down

DO NOT add serum at any point to these cells once the SCM is removed and the cells are exposed to trypsin

Resuspend the pellet in day 0 SFM and pipette cells up and down at minimal pipette force for ~ 20 times.

Plate the cells on a tissue culture treated T-80 flask in 12 ml of media

Feeding cells:

I see a considerable (~10-20%) cell death after 1 day of step-down. I therefore re-feed the cells the next day with day 0 SFM and then after every 2 days starting on 4^th day with the day 4 SFM.

Re-suspend the cells ~20 times with the pipette settings set to minimal speed. This is a key step to obtaining nice tight cell aggregates.

The day 10 SFM starts on day 10 and the clusters are fed every other day. I do not spin the cells in a centrifuge at any point. I allow them to settle in a 50 ml conical tube and pull off the supernatant once the aggregates have settled down (usually in a minute or so).

ICC

Immunocytochemistry

Wash cells 3 times at room temperature (5 minutes each ) in Dulbecco’s PBS containing calcium and magnesium.

Fix cells in fresh pre-warmed (37°C) 4% paraformaldehyde for 20-30 minutes at room temperature. (Gluteraldehyde is another fixative that may be used (0.25% to 4% in 1X PBS). It cross-links proteins and generally retains their antigenicity.)

2. Wash cells once at room temperature (5 minutes each) in Dulbecco’s PBS.

3. Preps can be permeabilized at this point using chilled 50% methanol in 1X PBS. Preps should be incubated in chilled methanol for 15 minutes. Cells can also be permeabilized in 0.2% (v/v) TritonX-100 in 1X PBS. Generally this is performed for up to 5 minutes at room temperature immediately following fixation. Following permeabilization, the cells are usually washed 3X (5 min each) in 1X PBS prior to the blocking step.

4. Block for 30 minutes at room temperature in PBS blocking buffer.

5. While the cells are blocking, set up a humid chamber for performing the antibody incubations. The chamber can consist of absorbent paper, soaked in water and a plastic storage container with a lid.

6. Dilute the primary antibody (or antibodies if performing multiple labeling) in PBS blocking buffer. Generally 50-100ul of diluted antibody solution per coverslip/slide. It is a good idea to make enough primary antibody solution for one more than the number of coverslips etc. Use PAP- Pen to minimize antibody volumes.

7. Add diluted primary antibody and incubate at 4°C overnight. This generally gives lesser background. However, you may incubate the primary antibody at 37°C for 1 hour or at room temperature for 3 hours.

8. Remove primary antibody solution and wash 3X in PBS (5 minutes each wash).

9. Dilute secondary antibody in PBS blocking buffer.

10. Incubate in secondary antibody in the humid incubation chamber for 1 h at 37°C.

11. Wash coverslips / labteks as before in PBS for 3X.

Add a drop of mounting fluid; MOWIOL or Vectashield, containing Hoechst 33342. Mounting fluid should contain 10µl of (a 10mg/ml stock) Hoechst 33342 as a nuclear counterstain in 1 ml of mounting fluid. Hoechst 33342 is a nuclear stain that binds to the minor groove of DNA (absorbance max is at 340nm and emission max is at 450nm). Alternate nuclear counterstain includes DAPI (10mg/ml stock in 1X PBS), which also binds to the minor groove of DNA (359nm and emission max 461nm) and propidium Iodide (10mg/ml stock in 1X PBS) that intercalates in the DNA (absorbance max 536nm and emission max 617nm).

12. Seal with nail paint.

13. View on fluorescence microscope or place in dark at 4°C for long-term storage. Note: it is strongly recommended to obtain an image of staining ASAP; paraformaldehyde fixation is reversible with time and fluorescent signals can fade off depending on the storage and anti-fade reagents used in the mounting fluid. Therefore, one cannot be certain that the observed antigen localization is correct if it has not been viewed immediately after mounting.

To prep live mounts

Unlike fixed samples, live cells, which are adherent to coverslips, do not tolerate washes in PBS without calcium or magnesium. It is therefore suggested to supplement PBS with 0.2mM CaCl2. This is a low enough calcium concentration that will not precipitate in the presence of phosphate salts. It is obvious to state that the permeabilization step be omitted. Mounting fluid should not be added to live cell studies.

Notes:

Fixation

Paraformaldehyde should be made fresh. The use of 4% paraformaldehyde as a fixative works for most antibodies and may be used up to 10% in some cases. A non-cross-linking fixative such as cold (-20°C) methanol can be used; due to its extraction of some lipids, methanol can destroy membranous organelles. PFA fixation does not have this effect on lipids and consequently better preserves organelle structure.

Controls

Secondary Antibody Alone

In order to control for the possibility of the secondary antibody cross-reacting with cells, it is necessary to set one coverslip aside and omit primary antibody incubation step. Incubate this control coverslip in PBS Blocking Buffer only for one or more hours. This control also helps eliminate signals that are due to autofluorescence of the cells, in the case of fluorescently tagged antibodies or endogenous peroxidase and phosphatase in the case of enzyme conjugated antibodies. If the background is high, it is recommended to analyze the cells without secondary antibody as well. This will help to determine whether the background is due to the secondary antibody or the cells themselves. If the cells show a lot of autofluorescence (esp. in the green channel), then use the proper controls to take the samples over to the META for emission fingerprinting.

Preimmune/Normal IgG

In addition to omitting the primary antibody, preimmune or “normal” IgG from the animal species in which the primary antibody was raised are sometimes incubated as a control. This control rules out the potential that any signal seen with the primary antibody is actually due to nonimmune IgG cross-reacting with antigen in the target cells.

Blocking

The PBS Blocking Buffer generally should contain normal serum from the host species that is used to generate the secondary antibodies. In theory, if there are any host IgG molecules that can cross-react with the cells being studied, then the unlabeled/non conjugated IgGs present in the normal serum will cross-react during the blocking step.

For antibodies that are raised against specific peptides, it would be a good idea to block these antibodies with the specific as well as non-specific peptides so as to achieve more assuring controls. To block antibodies with peptides, use 4X by weight, the amount of peptide to antibody.

Antibody (1mg/ml): 5 ml + Peptide (1mg/ml): 20 ml + PBS 1X: 75 ml à Incubate O/N on a rotator at 4°C. Next day spin @14K for 2’. Remove the top 95 ml, further dilute to appropriate working concentration and proceed for immunohistochemistry.

Solutions

Dulbecco’s PBS

139mM NaCl

2.7mM KCl

0.75mM CaCl2

0.48mM MgCl2-6H20

8.8mM Na2HPO4-H2O

1.48mM KH2PO4

PBS (without calcium and magnesium)
4% paraformaldehyde

Dissolve paraformaldehyde in pre-warmed Dulbecco’s PBS and then add one or two drops of 10N NaOH.

Blocking Buffer

4% normal donkey serum in PBS

note: 1-4% BSA (Fraction V) may also be used.

Recipe for MOWIOL

Tris 0.2 M, pH 8.5 (MW: 121.14) prepared by adding 580 mg Tris to 24 ml water.
Add 12 g of glycerol
Add 12 ml of dH₂O
Stir on a hotplate at no more than 55°C
Add 4.8g of PVA in small batches and stir till entire contents dissolve
Then add 2.5% (w/v) of DABCO
Cool to RT
Aliquot 1 ml into eppendorf tubes.
Freeze at –20 °C and store in dark.

Hoechst (345/478) may be added prior to use at 10-20mg / ml

Transwell

Cell migration: Transwells

Place desired number of transwells in conventional 24 well plate (I generally attempt to do 15 to a maximum of 18 at a time). Trypsinize PANC-1 cells (passage 4-8) and aliquot out 30,000 cells/½ ml/well. Put cells in DMEM containing 10% FCS and place in top of transwell. In addition to the transwells, plate 30,000 cells/½ ml in 24 well dish (as a control for trypsinization). * Shake the plate sideways after putting it in the incubator so that cells are evenly distributed on the transwell membrane and do not accumulate in the center (or edges).

Let cells attach for around 2 to 3 hours at 37°C.

1) In a new 24 well plate, place 400 µl media (minimal media with 0.05% gelatin (stock 2%) with appropriate potential migration factor(s) in bottom wells (in triplicates)). (Use 1000 µl BT tip to transfer media and don’t empty the tip completely so there are no bubbles introduced in the bottom well). Use appropriate controls:

positive control and minimal media with .05% gelatin alone

2) Remove media from cells that have been growing in transwells using 10µl BT tip. Trypsinize cells with 500 µl trypsin until cells round up (use control wells to determine this). Remove trypsin with 1000 ul BT tip. Move transwells to wells with potential migration factor(s) in 24-well plate. Move all transwells. Add 100 ul minimal media and 0.05% gelatin to upper transwell using 100 ul BT tip (take care that you do not introduce bubbles). Put membrans into well on a slate to prevent bubbles.

3) Make sure there are no bubbles under membrane!!!!!!

4) Incubate for 3 ½ hours.

5) Move transwell to an empty space in the 24-well plate.

5) Remove media from top of membrane and wipe top (only) of membrane with a cotton Q-tip (use ~4 Q-tips/membrane).

6) Start fixing cells. After that count cells in the bottom of the 24 well dish (wells that the membranes sat in during the 3 ½ hour incubation.

To fix cells

1) 4% paraformaldehyde in dulbeccos PBS- pH 7.4 ( 1 g paraformaldehyde in 25 ml DPBS with 2-3 drops of 10n NaOH - heat to 60C for a while, adjust pH to 7.4, use fresh) to top and bottom of transwell for 10 minutes.

2) Count cells in bottom of transwell.

3) Suck off paraformaldehyde in membranes and on bottom of well.

4) Add syto-61 in PBS with Ca/Mg (1/1000- stock is 5 mM, working solution is 5 uM, - keep in dark)(freezer storage box # 6 or 7), approx 400 ul in bottom of transwell, 100 ul top, 37°C X 20 minutes (Syto-61 is kept in freezer, stains nucleus). (Keep this stain in the dark). Also add 20 ul stock PI (10 mg/ml in DPBS)/10 ml of PBS.

5) Suck out dye- rinse c PBS once (Fill transwells with PBS from top and let drain down).

6) Using plain glass slides. Cut membrane with razor blade and put topside down, 2 membranes/slide, then put 1 or two drops vectashield on membrane add one cover slip for 2 membranes and seal with nail polish.

7) Count whole membrane under 10X objective (approximately 15 fields), use DS red filter (Rhodamine).

Original reference is Klepes, V.E., et al, Journal of Cell Science 114, 4185-5195 (2001).

Minimal Media for PANC-1 cells

For 100 ml of media

DMEM (low glucose) + F12 (1:1)

ITS (Gibco # 51300-044) (fridge)……………1000 ul

NIC (100 mM stock, 10 mM final)…1ml, fw =122.1 (1.2g/100 ml=.1M) or add powder directly to media 0.122g/100 ml

KGF (1000X) (10 ng/ul stock)………100 ul

BSA fatty acid free(1% final)(ICN # 152401) (fridge)…………..1.0 g

islet isolation

Mouse islet isolation

Euthanize mice with compressed CO₂ followed by cervical dislocation to ensure any discomfort to the animal. Submerge in 70% EtOH to disinfect. All of the following procedure, unless otherwise specified, is performed in asceptic conditions on ice.
Open the mouse to expose the spleen.
Pull out the spleen and the majority (~70%) of the pancreas attached to the spleen. (You may also pull out the mesenteric mass of the pancreas at this time).
Remove pancreas and put them directly into 20ml DMEM, in a 50 ml conical tube. Pool the pancreas from 3-6 mice. At this time make sure that there is no fat attached to the excised tissue (fat usually floats) and wash the tissue with cold DMEM medium for a couple of times.
Remove as much media as possible and transfer pancreas to a 10cm dish.
With scissors chop tissue to pieces around 2-5 mm3.
Transfer tissue to 7ml collagenase (3mg/ml) in a 15ml tube (this is generally enough for pancreas from 4-5 mice).
Seal tube with parafilm and shake vigorously in a 37˚C water bath for about 3-5min, continuing to shake gently for another 3-5 minutes or so or until the tissue is digested (no large visible chunks).
Add 2-3 ml FCS to inactivate Collagenase.
Spin For 3 min at 150xg.
Wash 2 x in RPMI.
Plate cells in non-tissue culture treated flasks in RPMI (containing 10% FBS).
Incubate at 37˚C for 3 days in RPMI (May need to Refeed inspect for exocrine and ductal cell attachment and death).
Spin down islet at 150xg for 3min.
Resuspend in CMRL 1066 + 10%FCS + Glutamine + penn/strep.
Plate islets on tissue culture treated dishes and maintain cells in SC CMRL.

ChIP

Chromatin immunoprecipitation

(obtained from Dr. Raghu, Mirmira, UVA)

Reagents:

ChIP sonication Buffer (1% Triton X-100, 0.1% Deoxycholate, 50 mM Tris 8.1, 150 mM NaCl, 5 mM EDTA):

10 ml 10% Triton X-100
1 ml 10% Deoxycholate
5 ml 1 M Tris-Cl pH 8.1
1 ml 0.5 M EDTA
3 ml 5 M NaCl
80 ml Water
Just before use, add 10 ul Aprotinin, 10 ul Leupeptin, and 5 ul PMSF to each 10 ml.

High Salt Wash Buffer (1% Triton X-100, 0.1% Deoxycholate, 50 mM Tris-8.1, 500 mM NaCl, 5 mM EDTA)
10 ml 10% Triton X-100
1 ml 10% Deoxycholate
5 ml 1M Tris-8.1
1 ml 0.5M EDTA
10 ml 5M NaCl
73 ml Water

LiCl Immune Complex Wash Buffer
25 ml 1M LiCl
5 ml 10% IGEPAL
5 ml 10% Deoxycholate
1 ml 1M Tris-8.1
200 ul 0.5M EDTA
64 ml Water

Protease inhibitors (add 10 ul of each to 10 ml of PBS or sonication buffer)
Leupeptin 2 mg/ml in water
Aprotinin 2 mg/ml in water
PMSF 0.2 M

5 M NaCl

1X TE Buffer (10mM Tris, 8.1, 1 mM EDTA)

0.5 M EDTA

1 M Tris-Cl, pH 6.8

Protein A/G Agarose (Santa Cruz)

Proteinase K (19 mg/ml, Boehringer Mannheim # 1964372)

10X proteinase K buffer

Elution Buffer (1% SDS, 0.1 M NaHCO3, 0.01 mg/ml Herring sperm DNA, 2ng/ml CMV beta Gal plasmid)

10 mg/ml Herring Sperm DNA

37% Formaldehyde (ACS reagent grade)

1.25 M glycine

Protocol: Generalized for many cell types, but may require optimization for specific cell types (tested with mPAC, bTC3, aTC, HEK, HeLa, NIH3T3).

For all the following steps, use the pipets that are specifically designated for ChIP use only.

Day 1:

1. To each 10 cm dish of cells, wash plate once with 10ml of PBS, then add 10 ml of Fresh PBS and add 270 ul of 37% formaldehyde, swirl gently to mix, and place at room temp 10 min.

2. At the end of the incubation, add 1 ml of 1.25 M glycine, swirl to mix.

3. Aspirate medium

4. wash plate with 10 ml cold PBS x 2. Aspirate PBS completely after the second wash
.

5. add 500 ul of cold PBS + protease inhibitors and scrape cells, collect in a 1.5 ml centrifuge tube. At this point you should pool three plates worth of cells together in the same tube (I suggest using a 2 ml siliconized eppendorf tubes for this purpose).

6. centrifuge at 2000 rpm for 2 min at 4 º C.

7. remove and discard PBS

8. add 600 ul of ChIP sonication buffer + protease inhibitors, and resuspend pellet (you can vortex vigorously at this point).

9. Place on ice for 10 min
.

10. Sonicate: We use a Misonix Sonicator (model S-300 sonicator with 2.5 in diameter cup horn and 8-place sample holder for sonicating multiple samples). The cup horn should be filled with an ice-water mixture. We recommend the following sonication settings:

Amplitude setting: 4

15 five second pulses with 15 second cool-down intervals between pulses. The shearing may be more efficient if the tubes can be placed at a 30-45 degree angle.

Alternatively, you can use a setting of 4 and deliver two 90 sec pulses with 1 min 45 s cool-down interval between pulses.

We found that the two protocols are very similar in shearing DNA to the 500-2000 bp range, however, the first protocol minimizes heat build-up.

11. Centrifuge at maximal setting at 4 C for 10-15 min.

12. Remove the supernatant into a fresh siliconized 1.5 ml eppendorf tube. This is the Whole Cell Extract (WCE), and can be stored at –80 C at this point, if desired.

13. Check protein levels by making a 1:10 dilution of a sample of the extract in water and doing a Bradford assay. Use 100 ug of WCE per antibody immunoprecipitation. If not enough extract is available, bring the solution up to 100 ug protein by adding purified/acetylated BSA.

14. Pipet 100 ug WCE into a fresh siliconized tube containing ChIP sonication buffer with protease inhibitors to a final volume of 1 ml.

15. Pipet 10% input into a 1.5 ml eppendorf tube (not siliconized, because it will dry faster after 70% EtOH wash—see later), and store at -20 C—you will need it later.

16. Add appropriate antibody volumes to each sample:

5 ul for histone antibodies from Upstate (acH3, acH4, 2meK4, 3meK4)

10 ul for histone antibody 1meK4

5 ul for Pdx-1 rabbit polyclonal antiserum, RNA polymerase antibody from Santa Cruz

2 ul for CTD RNA polymerase Ab

10 ul for RNA polymerase Ser2 and Ser5 Ab

17. Place the samples on a nutator in the cold room, and incubate overnight.

Day 2:

18. Resuspend Protein A agarose so that it forms a uniform suspension. Using a pipet tip with the end clipped off, add 45 ul of this suspension to each immunoprecipitation. Resuspend the protein A agarose each time before adding to the next sample, as it settles quickly.

19. Add 2 ul of a 10 mg/ml solution of herring sperm DNA

20. Place back on the nutator at 4 C for 1-2 h.

21. Centrifuge the samples at 4 C for 2 min at 2600 rpm.

22. Carefully remove the supernatant using a P-1000 and place it in a tube and label it “sample X—sup.” Place this at –20 C in case you need it later.

23. Add 1 ml of COLD ChIP buffer (no protease inhibitors), invert the sample to resuspend the resin, and centrifuge for 2 min. at 2600 rpm.

24.remove and discard the supernatant.

25. Wash X1 with High Salt buffer, X1 with LiCl buffer, and X1 with TE

26. Add 250 ul of Elution buffer to the resin, and place on a nutator at room temp. for 15-20 min.

27. Centrifuge at top speed to pellet the resin, remove the supernatant to a fresh tube.

28.Repeat the elution step (step 26), and combine the supernatants. (you can now discard the tube containing the resin pellet)

29. At this time, add 500 ul of elution buffer to the “10% input samples” from step 15.

30. Add 20 ul of 5 M NaCl to each sample, vortex to mix, and place in a 65 C bath for 3-4 h.

31. Add 1 ml of ROOM TEMP ethanol to each sample place at –20 C overnight.

Day 3:

32. Next day, spin the samples at top speed at 4 C for 15-20 min. to pellet the precipitated protein/DNA.

33. Aspirate off the supernatant, add 1 ml of ice cold 70% ethanol, spin again at 4 C for 5 min.

34. Aspirate off the sup, allow to air dry for 5-10 min.

35. Dissolve the pellet in 100 ul of TE.

36. Add 11 ul of 10X Proteinase K buffer, and 1 ul of a 19 mg/ml proteinase K solution.

37. Add 390 ul TE

38. Add 500 ul of PCIAA (phenol/chloroform/isoamyl alcohol)

39. Vortex for 30-60 sec and spin at max speed for 1 min.

40. Carefully remove the aqueous phase and transfer to a new tube.

41. Add 20 ul of 5 M NaCl, vortex to mix

42. Add 1 ml of room temp 100% EtOH

43. Incubate at -20 C overnight or -80 C for 1 h

44. Centrifuge at max speed for 20 min. at 4 C

45. Remove supernatant and add 1 ml of cold 70% EtOH

46. Centrifuge at max speed for 5 min at 4 C

47. Air dry tube

48.Resuspend in 100 ul of TE (Note: you may NOT “see” a pellet, but that’s OK!)

49. You’re ready for PCR.

Islet siRNA

Islet isolation and siRNA

Islets are maintained in RPMI 1640 medium containing P/S + 10% FBS with 25 mm Glucose.

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 m l 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 m l RLT buffer (RNA) OR 100 m l of 1X SDS sample buffer with out blue dye (protein) to the wells and keep. (50 m l of 2XSDS buffer + 50 m l 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

Coating cell culture plates

Glass dishes

Glass culture (Petri) dishes coated with gelatin will be used for the differentiation of hIPCs.

Cleaning and sterilization

Dissolve Terg-A-Zyme detergent (Fisher Scientific, Cat No. 04-322-11A) in distilled water (1% solution, 10 g per liter). Prepare the appropriate volume of the cleaning solution in the Nalgene Rectangular Tank.
Immerse the used 100-mm glass culture dishes (Kimble-Kontes, Cat No.23060-10020) in the cleaning solution. Let soak overnight so that residual cell material is dissolved. Make sure that all of the dishes are covered with solution.
Dish covers are normally not as soiled as the bottoms and can be cleaned separately (brief immersion into the cleaning solution and rinse).
Next day use the cellulose sponge to remove any cell debris from the dishes. Rinse repeatedly with distilled water. Perform the last rinse with double-distilled water.
Invert dishes and dry them upside down on lint-free paper.
Assemble dishes (cover and bottom), fix cover to bottom with small squares of autoclave paper and place in sterilization pouches (Fisher Scientific, Cat No.01-812-55) (three dishes per pouch).
Use P2 program (dry heat) to autoclave the dishes (Autoclave is in Room 4326).
Allow dishes to cool before use.

Note on cleaning: The plastic tank and cellulose sponges are not to be used for anything else than cell culture vessel cleaning. Tap water is to be avoided for rinsing.

Dish coating

Sterilized dishes can be coated with gelatin before use in FIPC culture

Warm up (37°C water bath) the stock 2% gelatin solution (Sigma-Aldrich, Cat No. G1393). When solution becomes clear, make up a 0.2% working solution (1 part of stock solution in 10 parts of cell-culture grade water).
Add 10 ml of the 0.2% gelatin solution per dish.
Incubate at 37°C in a humidified incubator for 1 hour.
Removed excess gelatin and let the dishes open in a vertical laminar flow hood to dry (10-15 min).
Dishes are now ready for seeding and can be used as any other cell culture vessel.

RNA quality: Basics of purity determination

RNA quality

Quantitation of RNA

The concentration of RNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. To ensure significance, readings should be greater than 0.15. An absorbance of 1 unit at 260 nm corresponds to 40 μg of RNA per ml (A260 = 1 => 40 μg/ml). This relation is valid only for measurements in water. Therefore, if it is necessary to dilute the RNA sample, this should be done in water. As discussed below, the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity. When measuring RNA samples, be certain that cuvettes are RNase-free, especially if the RNA is to be recovered after spectrophotometry. This can be accomplished by washing cuvettes with 0.1M NaOH, 1 mM EDTA followed by washing with RNase-free water. Use the buffer in which the RNA is diluted to zero the spectrophotometer. An example of the calculation involved in RNA quantitation is shown below:

Volume of RNA sample = 2.2 ml

Dilution = 10 μl of RNA sample + 490 μl distilled water (1/50 dilution).

Measure absorbance of diluted sample in a 1 ml cuvette (RNase-free).

A260 = 0.75

Concentration of RNA sample = 40 x A260 x dilution factor

= 40 x 0.75 x 50

= 1500 μg/ml

Total yield = concentration x volume of sample in milliliters

= 1500 μg/ml x 2.2 ml

= 3300 μg = 3.3 mg RNA

Purity of RNA

The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV, such as protein. However, the A260/A280 ratio is influenced considerably by pH. Since water is not buffered, the pH and the resulting A260/A280 ratio can vary greatly. Lower pH results in a lower A260/A280 ratio and reduced sensitivity to protein contamination. For accurate values, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure RNA has an A260/A280 ratio of 1.9–2.1 in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the spectrophotometer with the same solution. For determination of RNA concentration, dilution of the sample in water is recommended since the relationship between absorbance and concentration (A260 reading of 1 = 40 μg/ml RNA) is based on an extinction coefficient calculated for RNA in water.

RNA isolation and quantitative real-time PCR

Tissue samples were homogenized and frozen in Trizol (Invitrogen, Carlsbad, CA). RNA was isolated as per the manufacturers’ instructions, measured on ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and taken for reverse transcription / quantitative real-time pcr. First strand cDNA synthesis was carried out using ‘high capacity cDNA archive kit’ (Applied Biosystems, Foster City, CA). PCR was performed in 5 ml or 10 ml total volume in 96-well plates using cDNA prepared from 100 ng of total RNA on a 7500 FAST real time PCR cycler (Applied Biosystems, Foster City, CA). Primers and probes were Assay-on-Demand (Applied Biosystems, Foster City, CA). For estimation of fold-changes by qRT-PCR when the initial transcript levels were undetectable, the initial Ct value was assigned to be 38, which would lead to a possible underestimation of the actual fold-change. All qRT-PCR results were normalized to 18S (VIC-labeled) ribosomal RNA carried out in duplex reaction (with FAM labeled target gene probes) to correct for any differences in RNA input. Conventional pcr was carried out on reverse transcribed samples using AmpliTaq Gold (Applied Biosystems, Foster City, CA) and known / published primers (Primer sequences for b-actin and NeuroD are available from the authors upon request). All pcr reactions were analyzed after 35 cycles of amplification.

For microRNA detection, complete mouse miRNA panel (Applied Biosystems, Foster city, CA) including 283 miRNAs was used. Reverse transcription was carried out using mature miRNA-specific primer sets (Applied Biosystems, Foster City, CA) and microRNA reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed on Applied Biosystems 7500 FAST system using miRNA-specific taqman-based probe-primer sets (Applied Biosystems, Foster City, CA). All sample plates included positive, negative and endogenous controls supplied by the manufacturer in duplicate.

For transfection of antisense miRNAs or microRNA duplexes, siPORT NeoFX, a lipid based reagent (Ambion, Austin, TX) was used for transfection as per the manufacturers recommendations. Briefy, siPORT NeoFX was diluted in Opti-MEM 1 medium and incubated at room temperature for 10 min. MicroRNA inhibitors or mutant-anti-miRNA were diluted in Opti-MEM to a final concentration of 30 nM. Diluted RNA and diluted siPORT NeoFX were mixed by gentle pipetting and incubated at room temperature for 10 min. The RNA/siPORT NeoFX complexes were then distributed to each well and overlaid with cell suspension. Cells harvested after 4 or 6 days of transfection were taken for transcript analysis (neuroD and nkx2.2) and immunostaining.

Target prediction and cluster analysis

Since mammalian miRNAs are generally thought to recognize 3’UTR of target mRNA via partial complementarity, we used carefully designed computational approaches to predict mRNA targets for mammalian miRNAs. Two target search engines from Memorial Sloan-Kettering Cancer Center (http://www.microrna.org/), miRanda software and target analysis by PicTar (http://pictar.bio.nyu.edu/) were mainly used to confirm targets for specific miRNAs. Normalized data sets from realtime pcr analysis of miRNA expression profiles were taken as input data for bi-directional clustering. Bi-directional clustering is one of the most widely used algorithms to recognize patterns in datasets with similar expression profiles. Since functional modules of genes are generally regulated together, such modules can be identified from the similarity of their expression patterns in a bi-directional analysis. Two-way clustering was performed in MatLab^TM, using the Bioinformatics Tool-box (MatLab^TM v 7.0, R 14), which basically groups the samples with similar matching gene profiles together across the X-axis. Genes within these grouped samples that show similar expression patterns are grouped together along the Y-axis. Bi-directional clustering thus offers an important tool to assess closely related samples as well as similar gene expression pattern within these sample groups.

Cell adhesion assay

Cell adhesion

1) Dissolve/dilute coating substrate in ddH2O at 4 C. A common working dilution for the laminin-1 positive control and BSA (Sigma A8412) negative control is 40 ug/ml. For SN-peptide, use 20 uM (MW of SN-peptide is 2412) for plateau or 2.5 - 5 uM for half-maximal adhesion. For fragment E8 or laminin-1 plateau and half-maximal adhesion are usually achieved at 0.5 and 0.05 uM, respectively.

2) Add coating solution (100 ul/well) with multipipetter to wells of a 96 well tissue culture plate (Costar #3595), cover, and place at 4 C overnight. Coat in triplicate or quadruplicate.

3) Invert plate and shake out coating solution. Pull off remaining coating solution from each well with a yellow tip pipetter.

4) Dilute 7.5% BSA (Sigma A8412) to 1% in ddH2O. Add 100 ul/well with multipipetter, cover and place at 4 C for 4 hrs. Thaw 10 x trypsin/EDTA (then dilute to 1 x in PBS), warm PBS and serum-free medium.

5) In last 45 min of block, pull off medium from cells in T75 flask, and add serum-free medium. Replace in incubator for 30 min. Subsequently, pull off medium, wash with PBS, add 1 x trypsin/EDTA for 1 - 3 min, pull off released cells, wash flask with 20 ml of serum-free medium, pellet cells in 40 ml of serum-free medium, make up in 6 ml of serum-free medium and count (15 ul of suspended cells plus 15 ul of trypan blue; add 15 ul to each side of hemocytometer; cell#/ml = combined count from both sides x 104). Dilute cells to 2.0 x 105/ml in serum-free medium.

6) Invert plate and shake out BSA blocking solution. Pull off remaining blocking solution from each well with a yellow tip pipetter.

7) Pour cells in Reagent Reservoir (Costar # 4870), rock to suspend, remove 100 ul/well with multipipetter and add to wells. Repeat rock/resuspension prior to removing cell suspension for each row. Place in incubator for 30 - 60 min (37 C). 8) Examine plate in invert microscope. Photograph selected wells if desired. Invert plate gently onto an absorbent diaper pad. Pull off remaining cell solution from each well with pipetter.

9) With multipipetter, slowly add 100 ul/well of serum-free medium down the side of each well (tilt plate; PBS is not recommended for this wash). Invert plate gently onto an absorbent diaper pad. Pull off remaining wash solution from each well with pipetter.

10) Slowly add 100 ul/well of serum-free medium down the side of each well. Examine plate in invert microscope. Cells in BSA negative control wells should be rare (if not, repeat wash). Adherent cells in SN-peptide wells remain mainly rounded or slightly spread. An exception is M2 melanoma, which spreads rapidly on SN-peptide. Adherent cells in laminin-1 wells should be all spread. Invert plate gently onto an absorbent diaper pad. Pull off remaining wash solution from each well with pipetter.

11) With multipipetter, slowly add 100 ul/well of freshly diluted 1% glutaraldehyde in PBS. Fix for 10 min at room temp. Invert plate gently onto an absorbent diaper pad. Pull off remaining fix solution from each well with pipetter.

12) With multipipetter, add 100 ul/well of freshly filtered (use 0.2 um syringe filter) crystal violet (0.1% in ddH2O; Serva # 27335). Stain for 25 min at room temp. Invert plate onto an absorbent diaper pad, then wash plate gently by immersion in a plastic tray containing tap water. Invert plate onto an absorbent diaper pad. Pull off remaining wash from each well with pipetter. Reimmerse in fresh tap water. Invert plate onto an absorbent diaper pad. Pull off remaining wash from each well with pipetter. Repeat an additional time if required. Allow to dry for 5-10 min at room temp.

13) With multipipetter, add 50 ul/well of 0.5% Triton X-100 (diluted in ddH2O). Allow to solubilize overnight at room temp. in a drawer. Read at OD 595. BSA background should be less than 0.1 OD. Laminin value should be about 1.0 OD. Plateau SN-peptide value is usually 70-80% of laminin.

Matrigel invasion assay

Cell migration

Description
Matirgel is considered as basement membrane and generated from EHS sarcoma. Matrigel contains not only basement membrane components (collagens, laminin, and proteoglycans)but also matrix degrading enzymes/their inhibitors and growth factors. Invasion of tumor cells into Matrigel has been used to characterize involvement of ECM receptors and matrix degrading enzymes which play roles in tumor progression.

Procedure
1. Thaw Matrigel at 4C overnight.
2. Dilute Matrigel (5mg/ml to 1 mg/ml) in serum free-cold cell culture media (RPMI1640, EMEM, DMEM, etc).
3. Put 100 ul of the diluted matrigel into upper chamber of 24-well transwell
4. Incubate the transwell at 37C at least 4 to 5 h for gelling.
5. Harvest cells from tissue culture flasks by Trypsin/EDTA.
6. Wash the cells 3 times with culture media (RPMI1640, EMEM, DMEM etc)containing 1 % FBS.
7. Resuspend the cells in media containing 1% FBS at a density of 10^6 cells/ml.
8. Gently wash gelled matrigel with warmed serum free-culture media.
9. Put 100 ul of the cell suspension onto the matrigel.
10. lower chamber of the transwell is filled with 600 ul of culture media containing 5 ug/ml fibronectin, as an adhesive subtrate.
11. Incubate at 37C for 20 to 24 h.
12. Remove transwells from 24-well plates and stained with Diff-Quick solution.
13. Scrape off noninvaded cells on the top of the transwell with a cotton swab.
14. Count invaded cells under a light microscope.

Recipes
- Matrigel (Becton-dickinson)
- 24-transwell (Coster)
- Fibronectin(Sigma)\ - Diff-Quick staining solution (Fischer Scientific)

Single cell pcr

Protocol under revision. Will be available shortly.

siRNA

siRNA design rules

RNAi target selection rules:

Targeted regions on the cDNA sequence of a targeted gene should be located 50-100 nt downstream of the start codon (ATG).
Search for sequence motif AA(N₁₉)TT or NA(N₂₁), or NAR(N₁₇)YNN, where N is any nucleotide, R is purine (A, G) and Y is pyrimidine (C, U).
Avoid targeting introns, since RNAi only works in the cytoplasm and not within the nucleus.
Avoid sequences with > 50% G+C content.
Avoid stretches of 4 or more nucleotide repeats.
Avoid 5URT and 3UTR, although siRNAs targeting UTRs have successfully induced gene inhibition.
Avoid sequences that share a certain degree of homology with other related or unrelated genes.

How to obtain a cDNA sequence for target selection

Before finding a RNAi target on the gene of your interest, first you have to get its mRNA sequence or sequence accession number as some siRNA design tools can take accession number as input. It is recommended to use the gene's RefSeq from NCBI, since the RefSeq represents non-redundant, curated and validated sequences. RefSeq mRNA sequences have unique accession numbers which start with NM or XM, followed by 6 digits. For example, NM_123456 (curated mRNA sequence) or XM_0123456 (model mRNA sequence predicted by genome sequence analysis). There are several ways of querying RefSeq.

Search LocusLink by gene name or symbol at http://www.ncbi.nlm.nih.gov/LocusLink/. Once the locus of your gene is found, scroll down to the "NCBI Reference Sequence (RefSeq)" section and look for mRNA.
Search Entrez Gene at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene, and select the right gene of desired organism. Once the page for the gene is shown, scroll down to the "NCBI Reference Sequence (RefSeq)" and look for mRNA.
Search Nucleotide database using Entrez query tool at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide and use Entrez Limits settings to restrict your query to the RefSeq database only

select "RefSeq" from the "Only from" menu, this restricts the query to the RefSeq collection

select "mRNA" from the "Molecule" menu, this restricts the query to mRNA RefSeq records

Homology search
The RNAi targeted region on the mRNA sequence of a gene should not share significant homology with other genes or sequences in the genome, therefore, homology search is essential to minimize off-target effects. Although most siRNA design tools provide BLAST option, some simply use NCBI BLAST tools which sometimes are quite slow. Here are some BLAST tools for homology search.

NCBI Blast tool: Nucleotide-nucleotide BLAST (blastn) or Search for short, nearly exact matches

Blat tool on UCSC Genome Website http://genome.ucsc.edu/cgi-bin/hgBlat

Ensembl Blast http://www.ensembl.org/Multi/blastview

Examples of RNAi target selection

References

1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494-8.
2. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan 15;15(2):188-200.

Static stimulation

Static stimulation of Islet

Insulin secretion

Islets or in vitro derived islet-like cell clusters (ICCs) should be taken into 50 mm glass peri plates and handpicked under a stereo microscope. During handpicking, we prefer clusters with a size of 150 um diameter or smaller as these contain most viable cells and are easily picked up with a fine drawn capillary / pipette or even with a 200 ul pipette. Around 200 islet equivalents (IEs) are handpicked in each eppendorf tube. These are then incubated in RPMI 1640 medium without (control) or with the addition of either incretins (100 nm GLP-1 and 5 _m CCK) or NIC (10mm) for a period of 6 h (in some experiments ICCs were exposed to these agents for 3 d). After incubation of ICCs for 6 h, their ability to secrete insulin in response to glucose was examined by in vitro static stimulation. Briefly, ICCs are washed with PBS (1 mm CaCl2, 0.5 mm MgCl2, 26.7 mm NaHCO3, and 20 mm HEPES) containing 0.2% BSA (Sigma) and exposed in quadruplets to this basal (PBS and 2.8 mm glucose) or stimulated (PBS and 20 mm glucose) buffer for 1 h at 37 C.

At the end of the hour, cells were pelleted down by centrifugation, and the supernatant was assayed for the insulin released. After static stimulation, as described above, the insulin content of the ICCs was estimated after sonicating the ICC pellet in 200_l acid/ethanol (18 ml 10 m HCl/liter 70% ethanol). Insulin was extracted by incubating the cells overnight in acid ethanol at 4 C. Insulin concentrations were measured by RIA of the lysates.

Note: Though centrifugation may be carried out, I generally recommend allowing the islets / clusters to settle down (by gravity) and gently pulling out the supernatant from top.

MTT assay

MTT powder is available from Sigma (Cat # ). Prepare a stock of 5 mg/ml in PBS. Filter sterilize and store at 4°C in the dark. Make up small amounts and use within a 2-4 weeks max. If blue crystals precipitate out, refilter.

Other reagents

Media (preferably without phenol red), Isopropanol or Isopropanol/0.04 N HCl

I. Method for Adherent Cells

1. Plate cells in 96-well plates to desired density (we usually seed 500 to 2000 cells per well).

2. After growth for the desired period (usually 2-4 days) remove the all media using a multichannel / repeater pipette.

3. Add 100 ul RPMI (without phenol red): 10% MTT stock.

4. Place in incubator for 3 hours.

5. Add 100 ul isopropanol to each well.

6. Pipette up and down 2-3 times to dislodge bottom crystals.

7. Place on plate shaker for 20 minutes to dissolve crystals. Do not set speed very high.

8. Read in ELISA reader at 570-690 nm within 1 hour after adding isopropanol.

II. Method for Non Adherent Cells

1. Plate cells to desired density in microtiter plates. Use 100 ul media per wells.

2. Add 10 ul MTT stock to each well.

3. Incubate 3 hours.

4. Add 100 ul isopropanol/HCl (0.4 N) to each well.

5. Pipette up and down 2-3 times to dislodge bottom crystals.

6. Place on shaker for 20 minutes to dissolve crystals. Do not set speed very high.

7. Read in ELISA reader at 570-690 nm as soon as possible after it is finished shaking.

1. There are two advantages of using method I for adherent cells as opposed to the simpler method II. The first is that by using PRMI without phenol red you eliminate the need to acidify the medium with HCl. This increases the sensitivity of the assay approximately 20%. The second is that by changing media you eliminate serum proteins which can precipitate when the isopropanol is added.

2. The MTT in solution is not very stable as mentioned above.

3. The sensitivity of the assay varies between 200 and 3000 cells per well.

References Mossman, J. Immuno. Methods 65 (1983):55-63 Denizot, F. & Lang, R.J.Immunol. Methods 89 (1986):271-277

qRT-PCR

TaqMan based duplex PCR

Isolate total RNA using Trizol reagent (Sigma) or RNeasy kit (Qiagen) as per the manufacturers instructions. cDNA is prepared using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). PCR is performed in 5 ml reactions in 96-well plates using 50 or 100ng cDNA input (prepared from 50-100 ng of total RNA) and TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster City, CA). Primers and probes are Assay-on-Demand (Applied Biosystems, Foster City, CA). qRT-PCR data are normalized to 18S rRNA carried out using VIC-labeled probe in duplex reaction in every well to correct for any differences in RNA input.

After RNA isolation, please input the RNA concentrations and total volumes in the excel sheet here. Once you have entered these values, all other calculations will be automatically generated in the sheet. Follow instructions on the sheet to prepare your reverse transcription and pcr mastermixes.

After preparing the plate, centrifuge it in the 5810R plate centrifuge at 3000 rpm for 5 mins and then place it in the 7500 FAST real time pcr system next to it. Use the following program for running the plate.

Use the following Excel file for calculations of master mix for reverse transcription and PCR.

Islet size estimation

Islet size

The actual size / diameter of islets can be measured by using the Excel spreadsheet here. Scan upto 600 islets or ICAs from your plate after putting them on the counting grid and then enter the data for the size measured on ocular scale in the table. The actual size in mm will be displayed in the table / summary on the excel file.

Cell counting and basics

PANC1 Cell maintenance

Culture media: