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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.
This page was last edited 06/26/2003
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