TYPE 2 cell isolation from rat lungs.                            Click here to return to week 3

An important part of this experiment is making sure all solutions and materials are available.  Solution 1 and Solution 2 refer to certain mixed solutions that will be described later.

The first part involves the coating of cell culture plates with IgG. This is done so that macs and other contaminants will stick to the IgG but type two cells will not.

1. Weigh 7.5 mg of IfG to 15 ml of Tris Phos, mix and sterilize through a .22u filter.  Add ~4 ml to 100 mm tissue culture dishes.
2. Incubate for 2-3 hourss at 37 deg. C
3. rinse 5 times with 5 ml Sol 2.
4. Add 10 ml of dMEM w/o serum (+abs) and hold plates (in incubator on or up to 1 week)
5. When ready, remove medium and add cell suspension.

The second part involves the actual extraction of cells from the rat.

    Elastase is much more expensive than the rat itself.  If a mistake is made (punctured lung etc.) do another surgery rather than waste the elstase.  The elastase must be warmed so that it can dissolve.  It is a suspension.

1. Obtain specific pathogen free Sprague-Dawley rats weighing 180-200 grams.
2. Inject intraperitoneally with (mixed in the same syringe):
                            Heparin (1000U/ml stock)  250U/animal,  .25ml
                            Pentobartital (50 ml/ml stock) 37.5 ml/rat,  .75ml
3. Wipe fur and skin of ventral body with 70% ethanol
4. Incise skin in ventral midline from lower abdomen cephalad to neck.  Make midline cut through abdominal wall.  Cur major artery to exsanguinate animal.  Make neck incision to exposet the trachea.  Insert blunt needle in trachea via a trachea nick , and tie in place.  Make a hole on the diaphragm to let the lung collapse.  Open chest and remove anterior rib cage and thymus.
5. Make nick in left atrial appendage and right ventricle.  Advance blunt needle to main pulmonary artery via incision in right ventricle.  Perfuse pulmonary vasculature with iced solution1 until lungs are blood free (~50 ml).  The heart and lungs will be pale now.
6. Remove lungs with attached mediastinum (DO NOT BREAK OR PUNCTURE LUNG)
7. Lavage with approximately 100 ml of iced EGTA solution.  The lungs will have capacities varying from ~6ml to ~11 ml.  Push the solution in gently and suck out as much as possible.
8. Lavage with 40 ml of 37 C elastase (60 ml syring w/o plunger).  Place the lung in 37 C steril 175 ml of sol 2 in a steril beaker.   Incubate for 20 min in 37 C water bath.  The elastase sol. will distend the lung by gravity.   The lung will be "hanging" from the beaker into the solution with the syringe full of elastase.
9. Remove the lungs and cut lung tissue free of bronchi and jediastinal tissues.  Cut lung sections into 50 ml conocal tube with 4 ml of DNase 1 solution.  Mince with sharp, curved sterile scissors for about 5 min till the particles are smaller than the pippetter hole.
10. Transfer to flask.  Rinse tube with 4.5 ml of IgG free FBS (neutralize the elastase).  The cells will be bound to IgG plates so at this stage they need to be kept away from IgG.  Then rinse tube with Sol. 2 bringing total volume to 20 ml.  All solutions should be at 37 C.
11. Stir from 15 minutes in magnetic stirrer, no foaming.
12. Filter in succession through a 100 u filter.  Then connect a 37 u and 15 u sirenge filters together and filter through both of them.  Wash through filters with solution 2 bring total vol. to 45 ml.
13. Centrifuge cells (program 5) (1561 fpm, 20C, 8 min, 400 g,).
14. Pour the supernatant out.  Gentlly vortex the pellets.  Resuspend in 30 ml dMEM w/o FC serum that contains abs at 37 C.  Pipette up and down gently.
15. Transfer to IfG coated plates at 10 ml/100 mm plate.  Incubate for 1 hour at 37 C in 7.5% CO2 incubator.  Remove plates and tip back and forth 3 times and rinse gentrly to dislodge nonadherent cells (epithelial cell yield will decresase with time on the IgG plate over 1 hour.
16. Transfer suspended cells to 50 ml concical tube, count cells, and spin as above.  Resuspend in dMEM + FC serum at 1million cells/ml.
17. Incubate. At day 2, wash with PBS.  AECII devolop between day 2-7.
 
C.NEO EXPERIMENTAL PROCEDURES (Exp. 4)  
    Most of these procedures are modified from Go-Shau's Crypto assay procedures.

    1. Growth of C. neoformans.
a)C.neo C#d was cultured in Sabouraud's broth (1% neopeptone and 2% dextrose) for 3 days at 35 deg. C.
b) C3D was washed three times in sterile water and resuspended at 2*10EE4 orgs/ml.

    2. Infection of AM monolayers and CFU assays
1)After AM cultured for varous lengths of time, 50 uL of C3d suspension in culture medium (2*10EE4 orgs/ml) was added to each AM monolayer.
b) After incubation with C3D for 24 or 48 hrs, AM monolayers with .1% Triton X-100 to release intracellular organisms and agitated to resuspend the organisms.
c) The number of CFU were determined by plating serial dilutions of the lysates on Sabouraud's dextrose agar.
d) CFU were determined in quadruplicate for each experiment and are expressed as mean +/- SEM CFU/ml of culture medium.
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