Type II Cell Isolation                                                                                    FGB 3/00

Attempt to perform all steps using aseptic technique. See page 3 before beginning.

Thaw Calf Serum, 1000X P/S, 2 aliquots of Goat IgG, 1mg/ml DNase and 10X HBSS, 2X 50 mMTris, pH9.5.

Prepare the following: 1. SIP. 2. Inhibitor Soln. 3. DMEM/F12 + Elastase  (see below for recepies).

In hood, coat 8 bacteriological plates with 10 ml of 500 ug/ml IgG in 50 mM Tris base pH 9.5 for 3 hours at RT. Rinse 3X with sterile 1X PBS before use. Use Pronectin coated plates or coat  enough six or twelve well plates with Type I collagen (100 ul + 100 ml 1X PBS).

1. Euthanasia:  Pentobarbital:  0.5 ml IP.

2. Perfusion:  Open chest, clamp IVC, transect descending aorta, clamp SVC, tie off trachea with butterfly tubing inserted, perfuse lungs via RV with 10 ml ice cold soln. I, while intermittently inflating!  Remove lungs with heart, trachea and tube still attached to a sterile dish on ice.

3. Lavage:  Lavage lungs 5X with 5 ml aliquots of ice cold Soln I. Remove as much lavage fluid as possible after last pass. Discard, or, for macrophage isolation spin cells down for 20 minutes/ 2000 rpm's/4'C. (10cc syringe)

4. Cell dissociation: Instill each set of lungs with 15 ml of DMEM/F12 plus P/S, plus elastase (4.3 U/ml; Worthington) and DNase (1mg/60ml) while lungs are set in a 30 ml beaker. (four lungs requires 60 ml DMEM/F12, 1 mg DNase, 258 units elastase).  (10cc syringe)

Set beaker in a 37o water bath.  Incubate 35 min., recirculating every 5-10 min. 

5. Stop digest: Instill 10-15 ml ice cold DMEM/F12 plus Pen/Strep, plus 1 mg DNase, 10% calf serum, and 250 mg/100 ml trypsin inhibitor to stop digestion.

6. Dissection: On a sterile bacteriological plate on ice, remove the thymus, heart and any other tissue

7. Mince: on ice, cut up two sets of lungs into small pieces then mince for 25 min.

8. Repeat with other sets of lungs. Add all four sets of lungs to one trypsinizing flask on ice.

9. Trypsinize: Stir all four sets of lungs in trypsinizing flask for 10 minutes while on ice.

Note: keep cells on ice for all subsequent steps until plating.

10. Filter: Sequentially filter and wash cells through a 160um, 41um, and 15um Nitex filter (Tetko) into sterile 300ml, 300ml again, then 500ml beaker.

11. Wash: beakers and filters (as you go so don’t dry out) with a total of 300 ml DMEM/F12 + P/S.

12. Pellet: Spin cells @ 1,400 rpm/10 min/4o in six 50ml conical tubes.

13. Pool Cells: pool into 50ml, wash, pellet, then combine pellet in 8 ml of DMEM/F12 + p/s.

14. Percoll cells: with a 1.08 gm/ml gradient  to remove RBC. To four tubes add 7 ml percoll then two ml cells at 2000 rpm/20 min./RT/brake off (32.5 ml SIP + 18.5 ml DMEM).

15. Pool cells at interface then put cells on ice until wash.

16. Wash cells, immediately, with 40 ml DMEM + P/S. Repeat wash a second time.

17. Re suspend cells in 40 ml DMEM/F12, 10% FBS + P/S.

18. IgG panning: Rinse the 8 bacteriological plates (previously coated with IgG) with sterile 1X PBS then add 5 ml of cells to each plate.

Incubate 1hour/37oC/5% CO2 to plate Macrophages and fibroblasts.

19. Collect non-adherent cells: Tip plates back & forth while swirling then collect cells. Repeat 2X with 5 ml of Room Temperature DMEM/F12+ P/S per plate.

20. Pellet cells @ 1,400 rpm 10 min/4oC.

21. Wash: Re suspend pellets and pool in 40 ml and spin as above.

22. Re suspend cells: in 10 ml DMEM/F12 +P/S.

23. Count cells: Mix cells very well then aseptically remove 25 ul of cells and add to 100 ul of 0.4% Trypan blue (d.f.=5). Cells/ml=avg.# cells per square x (d.f.=5) x (10,000.).

24. Dilute cells:  dilute cells with DMEM/F12, 10% FBS plus P/S to 1 million cells/ ml and plate 2.5 ml (2.5 million cells) per well of a 6 well plate (or dilute cells to 0.7 million cells per ml and plate 1.5 ml (1 million cells) per well of a 12 well plate).

Note: Save 50ul for cytospin and dilute 1/10 for 1X10 5 cells/ml and spin it all.

25. Incubate for 16 hours at 37’C then wash wells 3X with 37’C DMEM/F12 + p/s (NO FBS)!

26. Aspirate last wash then add 2ml of media containing growth factors (or as appropriate).

27. Incubate 48 hours at 37’C then add <1ml Trizol per well shake for 5 minutes then pool the three wells/Treatment into two 1.5ml microfuge tubes to be pooled again during RNA isolation.

Sterile Equipment needed:                            Non-sterile Equipment:

Two sets of surgical tools each containing:                     Alcohol spray bottle,

toothed tweezers, tweezers, 2 curved iris                       paper towels, blue diapers, kim whipes,

scissors, 2 locking forceps, large scissors.                     gloves, pipette aid, plastic bag for animals,

Mincing equipment: 2 curved iris scissors,                      one dozen 15 and 50 ml conicals

two tweezers. 10 ml pipettes, 18 and 22g                      1-200 ul tips, P-200, 1.5 ml tubes needles, 19g butterfly, 5, 10cc  syringes,                                               silk thread extra sterile um filters

three 100 ml bottles, four 30 ml beakers,                       glass plate rack for cell re suspension.

two 300, one 500 ml beakers, 250 ml bottle,                Two bulb syringes w/ adapters (to inflate

a trypsinizing flask with stir bar,                         lungs)

bacteriologic plates, six or twelve well plates.

Pentobarbital, extra 15, 41, and 160 uM filters.

2X-100 ml beakers for Elastase and Inhibitor soln.

Reagents: 1mg/ml DNase, Elastase, P/S, Percoll, DMEM/F12, FBS, Calf Serum, Trypsin Inhibitor (powder), Goat IgG: 11.4 mg/ml IgG (12.5 mg) two-1.1 ml aliquots, Soln. I and II. sterile 1XPBS, 10 mM Tris Base, pH 9.5, 100 mM CaCl2, 100 mM MgCl2.

Before you begin: Set up the following: 10 cc syringe (6) a 5cc syringe with Pentobarbital, Blue diapers, paper towels, kim whipes, alcohol spray bottles, bag, surgical string, 50 ml conical tubes for Macs, 15ml conicals (4) for percoll, waste jar for lavage, sterile beakers with solutions I, Elastase and Inhibitor solutions on ice and 4 butterfly tubes.

 

 Inhibitor Soln:              DMEM/F12 + Elastase:            SIP:                       1.08 gradient:

89 ml DMEM/F12                   62 ml DMEM/F12                   45 ml Percoll         32.5 ml SIP

10 ml Calf Serum                      2.4 ml Elastase             5 ml 10X HBSS   18.5 ml DMEM

100 ul of 1000X P/S                65 ul of 1000X P/S                 

1.0 ml DNase (1 mg/ml)           1.1 ml DNase (1mg/ml)            0.5 mg/ml IgG:

250. mg Trypsin Inhibitor                                                          Two-1.1ml aliquots of 11.4 mg/ml

                                                50 uM Tris Base, pH 9.5          bring up to 50 ml with

                                                3.028g Tris Base,                     10 mM Tris Base, pH 9.5

                                                up to 500ml after pH, Aliquot into 50ml tubes.

 

Type II cell prep.:

The Day Before the Experiment:

1. Add 1ul/ml of 1000X P/S to four bottles of DMEM/F12.

2. Make 500 ml of DMEM/F12, 10% FBS +P/S. Add 50 ml of FBS to a fresh bottle of DMEM/F12.

3. Prepare soln. I and Soln. II (see below).

4. DNase Soln. Make 1 mg/ml DNase (need 2.1 ml/prep).

                        Soln I                                       Soln II

500 ml=           4.09 g NaCl                             500 ml of Soln I

                        0.186 g KCl                             9.5 ul of 100mM MgCl2

                        0.177 g Na2HPO4                   7.0 ul of 100mM CaCl2

                        0.450 g Dextrose

                        5.0 ml 1M HEPES

                        pH to 7.4, bring up to 500 ml, then 0.2um filter.