Am. J. Trot'. Med. Hyg., 29(6), 1980, pp. 1147—1149
copyright ©1980by The AmericanSocietyof TropicalMedicineand Hygiene
SEPARATION
OF STAGES
FALCIPARUM-INFECTED
OF PLASMODIUM
CELLS BY MEANS
OF A FLUORESCENCE-ACTIVATED
CELL SORTER
GRAHAM
V.
BROWN,*
FRANCIS
L.
BATTYE,
The Walter and Eliza Hall Institute
Royal Melbourne Hospital,
AND
RUSSELL
J. HOWARDt
of Medical Research, Post Office,
Victoria 3050, Australia
Abstract.
Plasmodium falciparum parasites from long-term in vitro culture have been
labeled with the DNA-binding dye Hoechst 33258. After labeling, parasitized cells have been
successfully analyzed and sorted, using a fluorescence-activated
cell sorter, into populations
of uninfected, singly infected, and multiply infected cells.
Malaria-infected
blood usually contains para
sites of different life-cycle stages, and the asyn
chrony is particularly marked during long-term in
vitro culture of Plasmodiumfalciparum.
Methods
of density separation of infected simian and rodent
blood have been used to prepare enriched popu
lations of cells containing mature parasites but
such methods have not proved successful for pu
rifying populations of either uninfected cells or
cells containing young parasites. ‘@
In this paper,
a recently described method for analysis and sort
ing of P. berghei-infected mouse blood on the ba
sis of DNA content,7 using a fluorescence-activat
ed cell sorter,8 has been modified to allow
separation of cells infected during long-term cul
ture of P. falciparum.
MATERIALS AND METHODS
Infected cells were obtained from long-term cul
tures of Papua New Guinean isolates of P. falci
parum
maintained
in vitro by the candle-jar
meth
od of Trager
and Jensen.9' ‘°
Cultures
are
routinely maintained as an 8% group A erythro
cyte suspension in RPMI-HEPES
medium con
taining 10% human group A serum and genta
micin 40 @g/ml.Prior to the labeling procedure,
the cell suspension was centrifuged for 10 mm at
350 x g, the supernatant was removed, and cells
Accepted
*
To
whom
14 June 1980.
correspondence
should
be
sent.
t Present address: Malaria Section, National Institute
of Allergy and Infectious Diseases, National Institutes
of Health, Bethesda, Maryland 20205.
were resuspended to the original volume in me
dium without serum or human tonicity phosphate
buffered saline [7 g NaCl, 2.5 g Na2HPO4 2H2O
and 0.625 g NaH2PO4@ 2H20 per liter (pH 7.3)]
(PBS).
The DNA-binding dye 33258 Hoechst was ob
tained from Riedel-de-Haen
AG.,
P.O. Box
1180, 3016 Seelze, West Germany. It was dis
solved just prior to use and kept in darkness
throughout
each experiment as previously de
scribed.7 Washed cells from culture (2 X 107/ml)
were incubated in medium or PBS containing
varying concentrations of dye (usual concentration
200 aim).
Incubation
was carried
out for 90 mm
to 2 hours in test tubes, in darkness, at 37°C.
Cells were then washed three times in PBS at 4°C
in darkness and samples were stored on ice prior
to analysis. Parasitemia was calculated as the ra
tio of infected erythrocytes to the total number of
red cells examined (5,000) in an area in the middle
of the smear with even red cell distribution. The
fluorescence-activated
cell sorter was operated
with a laser emission of 20 mW in the ultraviolet
(351.1 nm/363.8 nm) with low angle (1°—15°)
light
scatter and fluorescence signals simultaneously
detected for each cell. Further details of operating
conditions have been described previously.7 Cells
collected from the sorter were pelleted and thin
blood smears were made in the presence of fetal
calf serum before fixing in methanol and staining
with Giemsa stain. At least 100 sorted cells were
counted.
Analysis of low angle light scatter (an increasing
function of cell size)― confirmed the impression
gained on light microscopy of variation in red cell
size and morphology after long-term incubation
of cells at 37°C. For fluorescence analyses only
1147
1148
BROWN,
BATTYE,
AND
HOWARD
_@
SAMPLE
1—
NUMBER
OF
CELLS
FIGURE
1.
Fractions
(0, 1, 2, 3) sorted
following
particles of cell size, as indicated by their low an
gle light scatter, were analyzed. Smaller pieces of
debris and extracellular parasites were electroni
cally excluded.
After incubation with dye for 2 hours, the per
centage of fluorescent cells out of 100,000 ana
lyzed by the machine was on average 39% greater
than the percentage of parasitized cells calculated
from Giemsa stain of the original sample. This
may be explained by an underestimate of the par
asitized
cell number
(e.g.,
fragile
cells break
1
Percentage of malaria-infected cells in samples collected
after sorting by fluorescence-activated
cell sorter (sam
ples indicated in Figure 1)
no.0123Uninfected100%7%5%3%Singly
TypeofcellFraction
infected—93%95%3%2
or more nuclei———94%
(Fraction
by fluorescence-activated
0) consisted
entirely
cell sorter.
of uninfected
cells.
Sample 2 gave 97% infected cells containing a sin
gle nucleus. Sample 1, with cells of lower fluores
cence than the major cell peak, contained 93%
infected
cells which
were
all very
immature
par
asites, some containing only a minimum amount
of stained cytoplasm. Sampling of highly fluores
cent cells (Sample 3) isolated multiply infected
cells and schizonts.
dur
ing smearing and parasitized cells aggregate at the
tail of the smear where they are not counted).
There is a ‘background' fluorescence of 0.1%
when unlabeled normal red cells are analyzed by
the cell sorter. With samples kept on ice in dark
ness, analysis could be satisfactorily performed for
at least 5 hours after the labeling procedure.
Figure 1 shows the distribution of cell number
against fluorescence. There is one major and at
least one minor peak.
Following analysis, different fractions (Fig. 1)
were sorted and analyzed for parasite content
(Table 1). The nonfluorescent
cell population
TABLE
analysis
DISCUSSION
The fluorescence-activated
cell sorter has been
used to separate malaria-infected red cells from
noninfected
cells and to separate the infected cells
according to stageof development. The technique
allows the study of changes in infected cells by
stage of development and the study of pure non
infected cells. The method is limited by the num
ber of cells which can be sorted (3,000 per second)
and will only be used for critical experiments
where microtechniques
can be used, or long pe
riods of machine sorting time are warranted. In
creased yield can be obtained by preliminary con
centration, say of schizonts, using density gradient
separation (data not shown). The viability of cells
following this procedure has not been tested by
attempting to establish new cultures but cells in
fected with P. berghei have been found to be in
fective for mice after sorting by this method.7
At present there is no alternative method of sep
aration of uninfected cells from cells containing
immature parasites, and this technique could be
used to study morphology or metabolism or as the
basis of an in vitro invasion inhibition assay.
SEPARATION
OF MALARIA-INFECTED
of knobs on the infected erythrocyte surface after
long-term cultivation. Exp. Parasitol., 48: 213—
219.
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