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Lacrimal Experimental Protocol (4/4/94)

Day Before:

1. Prepare (6) 50 ml conicals each containing 50 ml of DMEM (high glucose) and gentamycin (50 µg/ml); place in incubator (90% O2, 10% CO2).

2. Thaw FBS (place at 4°C).

3. Place BMS at 4°C and LM at 4°C. If not planning to use gelled substrate, coat at 4 - 8 µg/well in 100 µl of dH2O overnight at 4°C.

Day of Experiment:

1. Thaw on ice STI , hyaluronidase , collagenase , DNase , dexamethasone , putrescine, ITS, glutathione, EGF

2. Weigh out 12.5 mg of ascorbic acid (to 1 ml with ddH2O later, then filter sterilize).

3. Make up 1 ml of STI in 100 ml of DMEM/gent. (do as (2) 50 ml); mix well and add 5 ml each to six 60 mm dishes; place in incubator.

4. Make enzyme solution mixing together 500 µl of hyaluronidase (Worthington 'HSEP'; stock 6980 U/ml), 500 µl of collagenase (Worthington; 1:1 mixture of types II and IV; stock 4000 U/ml), and 100 µl of DNase (stock 1000 U/ml) to 8.4 ml of DMEM/gent.

5. Prepare 40 ml of EDTA/HBSS consisting of: 4 ml of 10 x HBSS, 8 ml of 10 x EDTA, and 28 ml of tissue culture sterile water.

6. Make up DMEM/gent. plus goodies as follows (for SFMOM, delete FBS, increase DMEM/gent. to 28.5 ml and add 30 µl of 10 µg/ml bFGF (Sigma F9786): dexamethasone 30 µl of 10 µg/ml stock putrescine 300 µl of 100 mM stock [l-ascorbic acid 60 µl of 12.5 mg/ml stock]* ITS 360 µl of 1.25 mg/ml; 1.25 µg/ml stock glutathione 300 µl of 1 mg/ml stock EGF 30 µl of 50 µg/ml stock HEPES 456 µl of 1 M stock FBS 3 ml (heat deactivated) DMEM/gent. 25.5 ml (high glucose) *add just before use 7. Prepare DMEM/STI/20% FBS by mixing 40 ml of DMEM/STI/gent. with 10 ml of FBS.

8. Perfuse (3) male 4 wk Sprague Dawley rats (from NCI) with DMEM/gent. (have 100 ml made up) for 3 - 5 min until lung and liver are white.

9. Remove lacrimal glands and place in a 60 mm dish containing ice cold DMEM/STI/gent. Place dish on ice while second animal is dissected.

10. In hood, transfer to new DMEM/STI/gent, and cut away duct with scissors.

11. Transfer to a new dish and stab (25 guage needle) and inject each gland two times with 1 ml of media. Use needles to pull apart gland. Try to remove capsule.

12. Mince glands (1-4 mm2 pieces) with two #10 blade scapels, then transfer minced pieces into an autoclaved 25 ml plastic flask using a transfer pipet. Rinse dish with DMEM/STI/gent.

13. Allow pieces to settle, then pull off medium and add 3 transfer pipet volumes of HBSS/EDTA/gent.

14. Let settle, then draw off and add 8 ml of HBSS/EDTA/gent. Put top on flask and place in incubator on vortex at lowest setting (120 oscillations/min) for 15 min.

15. Coat wells of a 96 well plate with 0.18 mg/well of BMS (= 40 µl/well of #634 [10 mg/ml] diluted 180 µl up to 400 µl with cold DMEM; tube on ice; pre-chilled pipet tip); or 106 µl/well of #754 laminin [1.7 mg/ml]. For coating, plate is placed on ice, then is gelled at 37°C in incubator for 1 hr.

16. Let settle, remove supernatant and add 7 ml of DMEM/STI/gent. for 2 min.

17. Remove DMEM/STI/gent. and add 5 ml of enzyme mixture in DMEM/gent. Place on vortex for 15 min at 37°C.

18. Adjust Percoll by adding 1 ml of 10 x saline (1.5 M NaCl) to 9 ml of Percoll. Make up 10% (0.33 ml adjusted Percoll + 2.67 ml DMEM/STI/20% FBS), 30% (1 ml adjusted Percoll + 2 ml DMEM/STI/20% FBS) and 60% (2 ml adjusted Percoll + 1 ml DMEM/STI/20% FBS) Percoll. Pour a 60%/30%/10% gradient using a 1 ml pipet. Place in incubator.

19. Transfer solution to a 15 ml tube. Rinse out flask with DMEM/STI/gent. and add wash to 15 ml tube. Do 2 washes.

20. Spin cells at 500 rpm for 3 min (RT).

21. Discard supernatant, flick briefly to loosen pellet and add HBSS/EDTA/gent. to 12 ml. Invert to mix and spin for 3 min at 500 rpm.

22. Discard supernatant. Add 3 ml of HBSS/EDTA/gent. to pellet. Loosen pellet by gently drawing in and out with a transfer pipet; transfer to flask. Wash out tube with 2 ml of HBSS/EDTA/gent.; transfer to flask and make up to 8 ml.

23. Incubate at 37°C on vortex for 15 min.

24. In inhibition experiments, add 50 µg/well antibody to BMS for 1 hr at 37°C.

25. Transfer to 15 ml conical. Wash flask out two times with DMEM/STI/gent and spin at 500 rpm for 3 min.

26. Discard supernatant, loosen pellet and add 2 ml of enzyme solution. Transfer to flask and wash out tube with 3 ml of enzyme solution. Incubate flask on vortex at 37°C for 20 min.

27. Remove suspension and place in a 15 ml tube. Wash out flask with 20% FBS/DMEM/STI/gent. and mix by inversion 2 - 3 x. Spin at 500 rpm for 3 min.

28. Discard supernatant and suspend in 10 ml of 20% FBS/DMEM/STI/gent. Transfer to a 50 ml conical. Use 10 ml syringe to remove cells; remove needle.

28. Filter through 160 µm and 25 µm Nitex filters hooked up in tandem; filter into a 15 ml tube. Wash with 3 ml of 20% FBS/DMEM/STI/gent. Spin at 500 rpm for 3 min, then resuspend pellet in 6 ml of 20% FBS/DMEM/STI/gent.

29. Add very slowly to top of 60/30/10% Percoll gradient and spin at 500 rpm for 15 min.

30. If doing antibody inhibition experiments, remove antibody and wash BMS two times with warm DMEM/gent (gel) or sterile PBS (coated wells).

31. Cells reside at 60/30% interface. Use transfer pipet to pull off Percoll to this interface, then pull off cells and resuspend in 20% FBS/DMEM/STI/gent. Spin at 500 rpm for 3 min. Resuspend again in 20% FBS/DMEM/STI/gent. (14 ml) and spin to remove all Percoll.

32. Discard supernatant and make up in 8 ml of DMEM/gent. plus goodies. Take 15 µl of cell suspension plus 15 µl of trypan blue, mix gently then add 15 µl to each side of the hemocytometer. Count # of dead (blue) and living cells in grid using 10 x objective lens. Cell number in suspension is [2(Y) x 104/ml], where Y is number of cells in grid.

33. Add DMEM/gent. plus goodies, such that have 6 x 105 cells/ml. Plate cells with multipipettor at 91 µl/well (0.55 x 105 cells/well). Incubate overnight in 6% CO2 incubator.

Next Day

1. Place dispase and trypsin on ice.

2. Let carbachol warm up on bench.

3. Make up 6 ml of fresh DMEM/gent. plus goodies using DMEM/gent. from incubator.

4. Carefully pull off media from overnight culture and retain for cell count. Wash two times with 100 µl each of DMEM/gent. plus goodies (add with multipipettor. Add 75 µl of DMEM/gent. plus goodies and incubate for 100 min.

5. Weigh out 5.5 mg of carbachol. Put VIP stock on ice. Label two sets of T=0 tubes and two sets of T=100 min tubes.

6. 15 min before end of above incubation, make up stimulation medium. Make 10 µl of VIP stock to 100 µl of DMEM to give 10-6 M VIP. Make 5.5 mg of carbachol up in 3 ml of DMEM to give 10-2 M carbachol. Add 60 µl of 10-6 M VIP and 60 µl of 10-2 M carbachol to 6 ml of DMEM/gent. plus goodies to give stimulation medium.

7. Remove and retain media (ÔT=0 minÕ). Replace with 75 µl of stimulation medium using multipipettor. Incubate for 100 min at 37°C.

8. Spin T=0 media at setting 6 for 6 min on Eppendorf centrifuge. Retain supernatant and store at -70°C. Make up trypsin/EDTA/dispase by adding 0.9 ml of 10 x trypsin/EDTA to 3.6 ml of dispase; put at 37°C

9. After 100 min, remove and retain stimulation media. Add 100 µl/well of dispase/trypsin. Place plate on vortex at 37°C for 30 - 45 min.

10. Spin stimulation medium at setting 6 for 6 min on Ependorf, retain supernatant as T=100 and store at -70°C. Keep pellet on ice to combine with dispase/trypsin removed cells.

11. Loosen cells and pull off dispase/trypsin cell suspension by drawing up and down in pipet tip and place in tube containing cell pellet from above. Wash each well one time with 100 µl of DMEM and place in cell pellet tube. Spin at setting 6 for 6 min on Ependorf centrifuge. Pull off supernatant with aspirator and add 121.2 µl of DMEM. Vortex and store at -70°C.

Assay of Samples

A. Peroxidase Assay

1. Put media samples at 4°C to thaw. Cell pellet should be quickly refrozen and replaced at 4°C. Pull DAB from freezer to warm up. Put 1 U/µl peroxidase stock on ice.

2. Weigh out 0. 1 g of DAB; place in dark.

3. Make 2.7 ml of 1 M Tris, pH 8, to 20 ml giving 0.136 M Tris.

4. Add 5.4 µl of 30% H2O2 to 6 ml of water, and store on ice.

5. Turn on computer and set up template. Use kinetic (L1), automix on, wavelength 450 nm, run time 5:00 (5 min), read interval 0:10 (10 sec).

6. For a peroxidase standard curve, make 10 µl of peroxidase stock up to 1000 µl in DMEM to give a solution of 10 mU/µl. Make 100 µl of 10 mU/µl up to 1000 µl to give 1 mU/µl. Make 100 µl of 1 mU/µl to 1000 µl to give 0.1 mU/µl.

7. To 96 well plate (on ice) set up several blanks, a standard curve and T=0, T=100 and cell supernatant using Softmax. For blanks, use 50 µl of DMEM. For unknowns use 50 µl. Set up standard curve as follows (have a zero point [STD01]): STD02 5 mµ 0 µl DMEM 50 µl of 0.1 mµ/ml STD03 10 mµ 40 µl DMEM 10 µl of 1 mµ/ml STD04 50 mµ 0 µl DMEM 50 µl of 1 mµ/ml STD05 100 mµ 40 µl DMEM 10 µl of 10 mµ/ml 8. Add Tris to DAB and vortex to dissolve. Adjust pH to 7.0 with about 10 drops of 1 N NaOH. Draw up with a syringe and pass through a .2 µm filter into a reagent reservoir.

9. To blanks, standards or unknowns, use repeator pipet (setting 5) to add 125 µl/well of DAB solution. Place plate on UV max drawer, then with fresh tips and reservoir use repeator pipet (setting 1) to add 25 µl/well of H2O2 solution, and start reading immediately for 2 min with automix at 450 nm. After run, reset Vmax (usually 5 - 7 points).

10. Plot and reset Vmax to represent steepest part of curve (5 - 10 points). Slope for each standard curve should be similar.

B. DNA Assay

1. Make up Capillary Assay Solution consisting of 400 µl of 2.5 x modified TNE, 20 µl of Hoechst dye (1 mg/ml) and 580 µl of filtered dH2O. [For 2.5 x modified TNE, mix in a conical: 40 ml of 5 M NaCl, 1.25 ml of 1 M Tris pH 8, 0.25 ml of 0.5 M EDTA, pH to 7.4 with 5-7 drops of 1 M HCl, then make up to 50 ml with 5 M NaCl and pass through a 0.2 µm filter; for Hoechst dye, dissolve 10 mg of dye in 10 ml of molecular biology grade dH2O and pass through a 0.2 µm filter; prepare also filtered molecular biology grade dH2O; store 2.5 x TNE, Hoechst dye and dH2O together in a covered box at 4°C].

2. Make up calf thymus DNA standard solutions: [A] 10 µl of 1 mg/ml stock made up to 1000 µl in DMEM, [B] 30 µl of 'A' made up to 100 µl in DMEM, [C] 20 µl of 'A' made up to 100 µl of DMEM, [D] 10 µl of 'A' made up to 100 µl DMEM. Use 50 µl of 'D' for 50 ng, 50 µl of 'C' for 100 ng, 50 µl of 'B' for 150 ng, and 50 µl of 'A' for 500 ng. Spin 10 min at RT.

3. In ependorf tube, mix together 40 µl of 2.5 x mod. TNE, 12.5 µl of spun sample plus 37.5 µl DMEM, (or 50 µl standard made up above), and 10 µl of capillary assay solution, mix. Store temporarily in dark. For blank use 50 µl DMEM plus TNE and capillary assay solution. Vortex, then spin 2 min at RT.

4. Add 100 µl of blank to end of 100 µl capillary tube, keep tube horizontal and seal with putty material. Insert opposite end of tube into holder, and then place in minifluorometer. Zero with zero knob.

5. Insert capillary tube containing 100 ng of DNA and use scale knob to set to 25 (reads as .25 units per ng DNA). Insert and read other standards and unknowns. Make sure no fingerprints on capillary tube. Final DNA value is reading x 16 (4 x from instrument standardization; 4 x from dilution factor).

6. Combine data from A. and B. in Excel. Open a new worksheet. Type test substrates (ie. BMS or Ln) for each of 96 wells in column A. Type 'T=0' at top of column B, followed by Vmax values (start at B2). Do the same for 'T=100' (column C), 'Not Secreted' (column D) and 'Total (B+C+D)' (column E). To add B+C+D, type in E2 '=B2+C2+D2' and hit return. To apply this calculation to all other wells, click on E2 and block out E column, under edit go to 'fill down' and then hit return. To normalize to DNA values, copy and paste all of column A names to a lower portion of column A (ie A40). In B40, type in the µg DNA value for A2 (obtained by 16 x fluorometer reading). Fill in below all other DNA values and title column 'µg DNA'. Title three adjacent columns 'T0 (mU/µg DNA)', T100 (mU/µg DNA)', and 'Tot (mU/µg DNA),' respectively. Columns can be widened by dragging column boundary to right. To obtain normalized secretion values in mU/µg DNA, type in C40 '=(B2*0.3165)/B40' and hit return. To apply to all of column C, click in C40, block out column, under edit go to 'fill down' and hit return. For column D, use formula '=(D2*0.3165)/B40' and for column E '=(E2*0.3165)/B40'.

C. MTT Assay

1. Coat 96 well plate with 1/3 the amount BMS used on 48 well plate.

2. Add 1/3 the amount of cells used on a 48 well plate in 1/3 the volume.

3. Incubate cells for as long as experiment requires (overnight usually). Add reagents to be tested to the well (again 1/3 the amount in 1/3 the volume).

4. After incubation for time required by particular experiment, add enough media to the wells to make the final volume 100µl.

5. Add 10µl of MTT A-B Solution. Mix well by tapping sides of plate. Incubate for 4 hours.

6. Add 100µl of MTT Solution C to each well. Mix thoroughly by repeated pipetting with a multichannel pipettor until the black, fuzzy crystals on the bottom of the well have dissolved.

7. Within 1 hour measure, the absorbance on an ELISA plate reader. Use a test wavelength of 595 nm and a reference wavelength of 630 nm.

D. LDH Assay

1. Set up UVmax to read kinetically for 5 min with automix at 490 nm.

2. Dilute 2 ml of 1 M Tris, pH 8, in 10 ml of ddH2O and add 49 mg of L(+) lactate.

3. On 96 well plate aliquot 50 µl of t=0, t=100 or cell pellet supernatant. For blank, use DMEM (for cell pellet) and DMEM + goodies (for t=0 and t=100; since DMEM + goodies gives rise to a change in reaction product). Use 25 µl 'multi-enzyme lin-trol' (Sigma #M2266) diluted with 25 µl of DMEM as positive control.

4. Add 12.8 mg of a dry premix of INT (Sigma #I-8377), PMS (Sigma #P9625) and NAD (Sigma #N-7004) to Tris/lactate solution. Mix and wrap in foil. (Premix is 167 mg of INT, 43 mg of PMS and 431 mg of NAD.)

5. Place 96 well plate in UVmax and add 125 µl/well of Tris/lactate/INT/PMS/NAD.

6. Express values as % of total.

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