THE ANNA BEZOS FACS ASSAY updated 17-3-1995


This assay is done in 96 V-well microtitre plates


CONTROLS

	2 x Negative controls
	no primary antibody, medium only
	1 x Positive control
	known positive for the cells you are testing


NUMBER OF CELLS
	
	minimum 5 x 10E4 cells/well
	2.5 x 10E4 cells/well if using large cells e.g. MM170
	10 x 10E4 cells/well if using small cells e.g. Lymphocytes


MEDIUM

	PBS + 1% FCS  or PBS + 0.1% BSA
	FCS (foetal calf serum)
	BSA ( bovine serum albumin)
	Medium is used at 4oC


FLUORESCENT ANTIBODY
	
	 Fluorescein conjugated  FITC Sheep a Mouse Ig
	use at 1/20 to 1/30 dilution (diluted in medium)

FIXATIVE
	
	75ul/well PBS
	75ul/well 2% Paraformaldehyde



METHOD

	1. Count the cells to be used in the experiment
	2. Calculate the number you require (include your controls)
	3. Spin cells 1200rpm/5 minutes
	4. Discard supernatant
	5. Resuspend cells in cold PBS + 0.1% BSA in a volume to give 
	    100ul/well
	6. Plate out 100ul/well into V- well plate
	7. Spin plate at 4oC 1200rpm/3 minutes
	8. Flick off supernatant and vortex plate to resuspend pellet
	9. Add primary antibody (or PBS + 0.1% BSA for controls) at
	    50 - 100ul/well
	10. Incubate plate on ice 30-60 minutes
	    Add 50µl/well cold PBS + 0.1% BSA
	11. Spin plate at 4oC 1200rpm/3 minutes
	12. Flick off supernatant and vortex plate to resuspend pellet
	13. Wash cells by adding 100µl/well of cold PBS + 0/1% BSA	14. spin plate 1200rpm/3 minutes
	15. flick off supernatant
	16. vortex plate to resuspend pellet
	17. repeat step 13
	18. Add second antibody Sheep a Mouse Ig (including
	      controls) at 50µl/well
	      Dilution of 1/30 in cold PBS + 0.1% BSA
	19. Incubate plate on ice (in the dark) 30 minutes
	      Add 50µl/well of cold PBS + 0.1% BSA
	20. Spin plate at 4oC 1200rpm/3minutes
	21. Flick off supernatant and vortex plate to resuspend 
	      pellet
	22. Wash cells by adding 100µl/well of cold PBS + 0.1% BSA
	23. spin plate 1200rpm/3 minutes at 4oC
	24. flick off  supernatant
	25. vortex plate to resuspend pellet
	26. repeat step 20
	27. add fixative to the dry cell pellet
	      150µl/well
	28. cover the plate with foil to protect from light
	      store at 4oC until ready to read on FACScan when
	      the cells can be transferred to RT20 tubes