Phagocytosis
Fluorescent labeling of yeast
Resuspend 5 grams of yeast in 50 ml PBS in 100 ml flask
Put flask for 30 minutes in boiling water bath while stirring.
Wash 5 x with PBS and 2 x in PB.
Adjust the concentration to of particles to 109 particles/ml. Can now be frozen at -20°C.
For labeling, resuspend the pellet of 2 x 1010 particles in 20 ml Na2HPO4 (50 mM, pH 9.2)
Add 2 mg TRITC, incubate 30 minutes at 37°C on a rotary shaker.
Wash 2 x in Na2HPO4 (50 mM, pH 9.2) and 4 x in PB.
Freeze aliquots of 109 particles/ml at -20°C.
Phagocytosis assay
Grow cells for at least 5 generation times in HL5, the density not exceeding 5 x 106 cells/ml
Harvest 2 x 107 cells, wash 1 x in PB and resuspend in 1 ml PB.
Mix 100 ml cells (2 x 106 with 12 ml labelled yeast 1.2 x 107) in glass tubes.
Incubate for 0, 5, 10, 20, 30 minutes.
Stop with 1 ml cold PB.
Add 100 ml Trypan blue (2 mg/ml in 20 mM citrate, 150 mM NaCl, pH 4.5).
Mix and shake for 5 minutes.
Spin 3 minutes 500 x g.
Remove supernatant. Resuspend in 1 ml PB.
Read in fluorescence spectrofotometer at 544 nm excitation, 574 nm emission.
Include the following blanks:
For autofluorescence: 100 ml cells and 12 μl unlabeled yeast in 1 ml PB + Trypan blue.
For background, unquenched fluorescence, 12 μl labeled yeast in 1 ml PB + Trypan blue.
Standard curve:
0,2,5,10 μl labeled yeast in 1 ml PB.
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