PMKrein
15/05/01

1) Bring mice to laboratory in animal housing cages. Place in fume hood.
2) Expire mouse immediately prior to lavage by cervical dislocation method. (Refer to animal resources Standard operating procedures)
3) ???? serum?
4) Dampen animal fir with 70% EtOH.
5) Using scissors, make a small incision in the animal skin at the abdomen, tear skin around entire body, and peal skin upwards to expose thoracic cage and neck. Remove skin from forearms.
6) Secure animal on styrofoam panel.
7) Dissect tissue from neck to expose trachea.
8) Make a small incision in the trachea, to allow passage of 23 gauge lavage tube into trachea. DO NOT CUT TRACHEA ALL THE WAY THROUGH.
9) Cut a 1- 1.5 inch segment of 23 gauge tube, carefully pass a 23 gauge needle into the tubing.
10) Pass tube and needle into trachea, and stabilize using surgical sutures.
11) Load a 1cc syringe with .9cc sterile saline.
12) Place syringe in end of 23guage needle in trachea, using forceps to secure tube in trachea carefully inject saline into the animals lungs.
13) Aspirate saline by pulling barrel of syringe. Stabilize trachea with forceps throughout.
14) Remove syringe from needle, inject recovered lavage fluid to 15ml falcon tube on ice.
15) Repeat procedure 4 washes per animal.
16) Pool experimental group animal lavages on ice. Record total fluid collected on data sheet.
17) Clip 6 sections of lung (upper left, middle left, lower left and same from right) Store in labeled vials (formalin?). Mouse #, experimental, lung section, date)
18) Mix fluid well, remove 1ml, place into 1.5ml tube on ice. The remaining lavage should be centrifuged at 1200rpm for 15 minutes with brake.
19) While the fluid is spinning, prepare a 1:10 dilution of lavage fluid by mixing 10µl of lavage fluid with 80 l RPMI + 10 l Trypan blue. Load 10 l into each chamber of the hemocytometer. Obtain cell concentration by counting five squares of the hemocytometer. Maintain two counts, one viable, one non-viable.
20) Calculate and record cell concentration as follows:
(Total cells counted/5 squares counted) x 10 x 4000 = cells per ml
Calculate and record total cell number by multiplying "cells per ml" by the volume of recoded (less 1ml fraction).
Cell viability will be recorded as percent viable:
(viable cells / total cells) x 100
20) While lavage is spinning, label tubes for freezing lavage sup. Prepare the following:
a.) NUNC 1.8ml cryovials. Label tubes with:
Experimental condition
BALF
Date

21) Remove lavage from centrifuge, pour sup. into Falcon tube on ice.

22) Resuspend cell pellet in 5ml 37 C RPMI + 1%penicillin/streptomycin (P/S).

23) Add sufficient RPMI to bring cell concentration to 2.0x106 cell/ml. Aliquot 2ml (therefore 4.0x 106 cells) into each well of a 6 well plate. Place into incubator for 45 minutes to allow Macrophage to adhere.

24) Aliquot 1.5ml BALF into NUNC tubes until all is stored.

25) Place all aliquoted sup. into labeled Sarsted box, immediately place into -70 C freezer.

26) Label 6 microscopeslides with the following information using a #2 pencil:
experimental condition
BALF
Date
Set up slides with cytofunnels in cytospin.

27) Compute volume needed for 50,000 cells per slide for each slide.
(Y x 104cells/ml) / (5x104cells/slide) x 1000µl/ml = X l/slide
where X equals µl to give 50,000 cells/slide.
and Y equals cell concentration from Step 6.
Multiply X l/slide from above by 6. Remove this volume from 1ml aliquot.
bring total volume to 3ml with RPMI+P/S in separate tube. Mix well then load 500µl into each cytofunnel.

28) Centrifuge slides in cytospin at 600rpm for 10 minutes

29) Remove all slides from the cytospin apparatus discard funnels, and allow to air dry at least two hours. After dry, stain one slide within 24 hours with Hemacolor stains as follows:
25 seconds in fixative #1
15 seconds in eosin Y (red) #2
15 seconds in thiazine (blue) #3
rinse in distilled water (rinse BACK side of slide)
Dry, mount cover slip with entallen.

All remaining slides are fixed. At least two slides should be fixed in each of the following ways: Methanol, Acetone, Formalin. Indicate on the slide with #2 pencil how slide was fixed.