MIGRATION STUDIES ON SHEEP AFFERENT LYMPH  Peta O'Connells Method
	
	Monoclonal Antibodies and Fluorescent Conjugates

Cells expressing sheep immunoglobulin were identified by direct
immunofluorescence using a 1:40 dilution of fluorescein conjugated, anti-sheep
immunoglobulin, affinity isolated fraction (Silenus Laboratories, Melbourne).
All other cell populations were identified using indirect immunofluorescence.
Murine monoclonal antibodies (mAb) directed against the sheep CD4, CD5 and T19
(gd T cells) antigens were supplied as a generous gift by Dr Charles R. Mackay,
Basel Institute of Immunology, Basel, Switzerland. Murine mAb directed at the
following sheep antigens were obtained from Dr. Mal R. Brandon, Centre for
Animal Biotechnology, School of Veterinary Science, University of Melbourne. MHC
I, CD45 (LCA), MHC II (28.1, 37.68, 38.27, 42.2O, 49.1), CD1c and CD8. Four
unique subtypes of ovine MHC II may be recognised (28.1, 37.68, 38.27 & 42.20).
All four subtypes are recognised by the mAb MHC II (49.1). A 1:80 dilution of
fluorescein conjugated sheep anti-mouse, F(abŐ)2 affinity isolated fraction
(Silenus) was used to fluorescently label the mAb.

	Immunofluorescent Staining

Lymph samples were spun to a pellet, the supernatant was discarded and red blood
cells were removed by osmotic lysis if required. The white cell pellet was
washed 3 times with phosphate buffered saline (PBS) (Appendix 1.1) and then
resuspended to 2 x 107 cells/mL in PBS containing 2% bovine serum albumin (BSA)
(CSL) and 0.1% NaN3 (BDH). 2 x 106 cells were incubated with 50 mL of mAb for 30
minutes at room temperature, followed by 3 washes in PBS/BSA/NaN3. The cells
were then incubated with 50 mL of fluorescent sheep anti-mouse conjugate. The
cells were washed a further 3 times in PBS/BSA/NaN3 and then fixed with 150 mL
of FACS Fixative (Appendix 1.3). Stained cell preparations were analysed by flow
cytometry (EPICS 741, Coulter Electronics Ltd.). Langerhans cells were
identified by CD1c positivity and by their forward and 90o scatter profiles,
whilst other cell populations were identified as the percentage of positive
cells.