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Induction of Differentiation and Cellular Manipulation of Human Myeloid HL-60 Leukemia Cells
|Induction of Differentiation and
Cellular Manipulation of Human
Myeloid HL-60 Leukemia Cells
The human myeloid HL-60 leukemia cell line has
enjoyed widespread use in studies of the molecular
mechanisms involved in the control of cell growth,
differentiation, and apoptosis. Following stimulation
with specific agents, these cells acquire a granulocytic
(Collins et al.
, 1980), monocytic, or macrophage-like
phenotype (Murao et al.
, 1983). The maturation of these
precursor cells into their mature progeny occurs at a
high percentage and with some degree of predictable
temporal uniformity. The acquisition of mature phenotypes
can be demonstrated by a variety of differentiation
markers. These include enzymatic markers
such as an increase in the activities of nonspecific
esterase or lysozyme; morphological changes such as
the appearance of segmented nuclei, which are characteristic
of a granulocyte-like phenotype (Collins et al.
, 1980); or other markers such as cell attachment
and spreading on culture dishes or increased phagocytosis,
which are indicators of a macrophage phenotype
(Tonetti et al.
, 1994). Additionally, there are a
variety of well-characterized immunological markers,
some of which are lineage and/or stage specific. The
use of these markers and forceful expression of specific
cDNAs or inhibition of a particular gene's expression
by pertinent antisense oligonucleotides has allowed
for the identification of the temporal order of
many components involved in the signal transduction
processes that lead to HL-60 cell differentiation
(Semizarov et al.
, 1998; Laouar et al.
, 1999; Xie et al.
II. MATERIALS AND
HL-60 cells (Cat. No. CCL-240) are available from
the American Type Culture Collection (ATCC).
Culture medium RPMI 1640 (Cat. No. 11875-093),
penicillin-streptomycin-glutamine (Cat. No. 10378-
016), geneticin (Cat. No. 10131-035), and trypsin-
EDTA (Cat. No. 15400-054) are from Invitrogen
Life Technologies. Phorbol 12-myristate 13-acetate
(PMA; Cat. No. P-1680) is from L.C. Laboratories,
Inc. All-trans retinoic acid (ATRA; Cat. No. R-2625),
L-0Mysophosphatidylcholine (Cat. No. L-4129),
paraformaldehyde (Cat. No. P-1648), bovine serum
albumin (BSA) fraction V (Cat. No. A-7906), anti-cdllb
(Cat. No. C-0051), transferrin (Cat. No. T-2036),
and insulin (Cat. No. 1-6634) are available from
Sigma Chemical Co. 1α,25-Dihydroxy-vitamin D3
vitD3; Cat. No. 679101] and hygromycin B
(Cat. No. 400050) are from CalBiochem. PolyMount
solution (Cat. No. 16866), 1.72-µm fluoresbrite beads
(Cat. No. 17687), and hydroethidine (Cat. No. 17084) are
from Polysciences Inc. 4',6-Diamidine-2'-phenylindole
dihydrochloride (DAPI; Cat. No. 236276) is available
from Boehringer Manneheim Biochemicals. Lab-Tek
chamber slides (eight-well, Cat. No. 178599) are from
Nunc, Inc. Fetal bovine serum (FBS; Cat. No. SH30071)
is from HyClone. Dimethyl sulfoxide (DMSO)
molecular biology grade (Cat. No. BP231-100) is from
Fisher. Antibodies against CD14 (Cat. No. 30541A) and
HLA-DR (Cat. No. 555562) are available from BD
Pharmingen. Antibody against human glyceraldehde-3-phosphate dehydrogenase (GAPDH, Cat. No.
CR1093SP) is from Cortex Biochem. Antibody against
actin (Cat. No. sc-1615) is from Santa Cruz Biotech.
Vector pRL-null (Cat. No. E227A) is from Promega,
and pIRES2-EGFP (Cat. No. 6029-1) is from BD
A. Cell Growth and Differentiation Induction
- Culture medium: Supplement RPMI 1640 culture
medium with penicillin-streptomycin (100µg/ml),
L-glutamine (200mM), and 10% FBS. If cells are to
be used for viable immunostaining, inactivate serum
complement by heat inactivation (65°C for 15 min followed
by slow cooling to room temperature). Culture
medium can be stored at 4°C for several months.
- Differentiation inducer stock solutions: Dissolve
PMA, 1,25-(OH)2vitD3, and ATRA in DMSO at a concentration
of 1 mg/ml. Store small aliquots in sterile
microcentrifuge tubes at-70°C and avoid repeated
- Differentiation inducer working solutions: The most
effective concentration for the induction of differentiation
depends on the potency of individual lots of the
inducer and the endogenous sensitivity of the HL-60
cells being used. Examples of appropriate concentrations
for some common inducers of HL-60 differentiation
are shown in Table I. For new chemical inducers,
a series of concentrations of the inducer should be
tested, and conditions that impart an inhibition of cell
multiplication and the appearance of one or more
myelomonocytic differentiation markers should be tested further. For many differentiation inducers, cells
will exhibit maturation markers at inducer concentrations
that inhibit about 50% or greater of cell multiplication
- Cell culture: The HL-60 cell line has a population
doubling time of 20-24 h. Cell density should be maintained
between 2 × 104 and 1 × 106 cells/ml with
replacement of medium at high densities by pelleting
of the cells by centrifugation if medium becomes acidified.
Cultures should be maintained in either petri
dishes or tissue culture plates at 37°C in an 8% CO2,
95% water-humidified atmosphere.
- Collect the cells and obtain cell density by hemocytometer
chamber counting. Pellet the cells by gentle
(250g) centrifugation. Resuspend in fresh growth
medium and plate the cells at 2-10 × 104 cells/ml in
culture dishes. Allow the cells to recover for several
hours prior to addition of the inducing agent.
- Dilute the differentiation-inducing agent in a
minimal volume of culture medium and add to the
experimental plates. Be sure to include a control
culture to which the solution vehicle alone is added.
- Culture cells at 37°C in an 8% CO2 humidified
- The appearance of differentiation markers occurs
as early as 12h and up to 6 days after treatment,
depending on the concentration of the inducing agent
and the marker to be assessed. To evaluate the phenotypic
changes associated with cell differentiation, a
series of morphological, biochemical, and immunological
assays are performed (see Table II). We recommend that two to three different assays be used to
determine whether the cells acquire a granulocytic,
monocytic, or macrophage-like phenotype. Many
of the biochemical or histochemical markers can be
assessed using commercially available kits.
B. Cell Adhesion and Spreading Assay
Phosphate-buffered saline (PBS)
|FIGURE 1 Bright-field microscopy
images representing an
example of HL-60
cells either untreated in suspension (A) or
attached and spread following a 3-day
treatment with 10nM PMA (B).
: To make 1 liter of
stock solution (10×), dissolve 80.0 g of NaCl, 2.0 g of
KCl, 2.0 g of KH2
, and 11.4 g of Na2
(Milli-Q) water and sterilize by autoclaving.
Dilute to l× in water.
- Treat HL-60 cells cultured in bacteriological petri
dishes (see Section IV) with PMA and incubate at 37°C in an 8% CO2, humidified atmosphere for 2 days.
- Collect unattached cells by gentle pipetting and
wash petri dishes twice with PBS, combining the
washes with the initially collected cells.
- Recover the remaining attached cells by treatment
for 10rain with PBS supplemented with 0.05%
trypsin-EDTA followed by forceful pipetting and/or
use of a rubber policeman.
- Count the number of attached and unattached
cells using a hemocytometer chamber and calculate the
percentage of attached cells as a function of the total
recovered cell number.
- In parallel, determine the number of spread cells
by counting flattened cells (Fig. 1) in several microscopic
fields of view. Count at least 200 cells/data
point; the percentage cell spreading = (number of
spread cells/total number of adherent cells) × 100.
C. Phagocytosis Assay
- Fluorescent beads: Sterilize and opsonize fluoresbrite
beads by first adding three drops of beads to
10ml of 70% ethanol. Mix and agitate at room temperature
for 20min. Recover the beads by centrifugation
(400g) and wash twice with PBS. After the second
wash, add 5 ml of RPMI 1640 supplemented with 1 ml
FBS (not heat inactivated). Mix and incubate overnight
at 37°C with slow agitation. Pellet the beads at 1000g and resuspend in 1 ml of culture medium at a concentration
of 4 × 108 beads/ml.
- Working solutions: Prepare the following solutions
using PBS as solvent: 4% paraformaldehyde,
10 µg/ml L-α-lysophosphatidylcholine, 0.1 µg/ml DAPI,
and 10 µg/ml hydroethidine.
- Plate 1.2 × 104 cells in 400µl of growth medium
into each well of an eight-well Lab-Tek chamber slide.
Treat the cells with PMA or other inducer as described
- After 30h, add 100µl of the bead suspension to
- Incubate for an additional 18h, gently remove
the growth medium, and air dry the cells for 30 s using
a heated slide dryer.
- Fix the cells by adding 100µl of 4% paraformaldehyde
to each well for 20min and then washing
each well several times with PBS.
- Add 100 µl of L-α-lysophosphatidylcholine solution
to each well for 20min to permeabilize the cells,
followed by several washes with PBS.
- Cell nuclei are stained by incubation with DAPI
for 5 min. Remove excess stain by washing with PBS
and then counterstain the cytoplasm by incubating
with hydroethidine for 10 min.
- After three washes in PBS, remove the well partitions
and mount the slide with Poly-Mount or other
mounting medium and a coverslip.
- Examine the cells using a fluorescent microscope
equipped with a DAPI/UV-range filter set. DAPIstained
nuclei should appear blue, the stained cytoplasm
red, and the fluoresbrite beads as small blue
dots. A cell is considered positive for phagocytosis
activity if it has engulfed more than 20 beads. Count
>200 cells per point.
- PBSA: dissolve BSA (Cohn fraction V) to 1% in
- Primary antibodies: Numerous monoclonal and
polyclonal antibodies are available to characterize the
phenotype of mature myeloid cells. These include but
are not limited to the Mac-1/antiCDllb, CD14, or class
II HLA-DR antigens. A more categorical listing is available
other/01-81024-3.pdf). These are available from
many commercial sources (e.g., Immunotech, Coulter
Immunology, Santa Cruz, Sigma, Pharmingen) and
should be prepared and used according to the manufacturer's
recommendations. For a new antibody, it is
often prudent to perform a dilution series with both
positive and negative controls to select an antibody
concentration that provides a positive signal with a
low background. For semiquantitative ratio imaging,
useful standard antibodies are against GAPDH (sheep
polyclonal, Cortex Biochem.) or actin (goat polyclonal,
Santa Cruz). All antibody stock solutions are created
by dilution in PBSA.
- Secondary antibodies: There are numerous sources
of labeled secondary antibodies. If one is to perform
multiantigen imaging, it is critical that the secondary
antibodies have been cross-absorbed against the
other host organisms antibody repertoire. Such crossabsorbed
antibodies are available tagged to a variety
of fluorophores from both Jackson Immunoresearch
and Molecular Probes. Stock solutions of these secondary
antibodies are prepared at 1 mg/ml in 50%
glycerol/50% PBS. Store aliquots of the stock solution
at-70°C. Prepare a working solution of the secondary
antibody by diluting from 1:50 to 1:500 in PBSA using
the furthest dilution that still provides significant
- Microscopy: A microscope equipped with multiple
filter sets and/or cubes is required to visualize the
fluorescence signal from multiple fluorophores. If one
is to perform semiquantitative ratio imaging using
multiple fluorescent signals, it is useful to have either
an automated cube turret or automated excitation and
emission filter wheels and a multipass dichroic cube. Both this hardware and SlideBook software for processing
such images are available from Intelligent
Imaging Innovations (Denver, CO).
- Flow cytometery: For the assessment of marker
induction in granulocytic differentiation, flow cytometry
has many advantages, particularly regarding the
stastical significance attainable by scanning larger cell
populations. However, in the case of monocytic and
macrophage differentiation, significant clumping of
cells is unavoidable and may lead to artifactual results.
We therefore recommend fluorescence microscopy for
such experiments and the examination of at least 200
cells per data point.
Reactions are performed on ice using either microcentrifuge
tubes or, to facilitate the handling of numerous
samples simultaneously, V-bottom 96-well plates.
E. Transfection and Establishment of
Stable Cell Lines
- Recover the cells by centrifugation and resuspend
in PBS at 2 × 106/ml; add 200µl of the cells for
each collection of antibodies to a well of the microtiter
- Centrifuge the plate (400g), remove the supernatant
gently by inversion, and fix the cells by incubation
with 100µl of 4% paraformaldehyde for 15min.
Wash twice with PBS.
- If the antigen to be visualized is not on the
outside of the cell membrane, permeabilize the cells by
the addition of either L-α-lysophosphatidylcholine
solution or 0.1% Triton X-100 in PBS for 5min. Wash
the cells three times with PBS.
- Resuspend the cells in 100 µl of PBSA and allow
for blocking of nonspecific sites by incubation for
15min. Recover cells by centrifugation and mix the
cells with 100µl of diluted primary antibody or
mixture of antibodies (or a dilution series may be used)
and incubate for 45 min at room temperature.
- After washing the cells twice with PBS, add
100µl of diluted secondary antibodies and continue
incubating for 30min.
- Wash the cells three times with PBS and then
mount onto microscope slides using a small volume
of a mounting solution. Overlay with a cover glass
and seal with nail polish. Examine by fluorescence
- Most chemical approaches (e.g., charged lipids)
for the transfection of HL-60 cells produce very poor
results. Better transfection is achieved using electroporation, particularly with a square-wave device, and
to a lesser degree by an exponential-decay device.
We use a BTX T820 square-wave electroporator and
achieve maximally 10-20% transfection rates.
- For analysis of promoter activities, either CAT
assays or luciferase activities can be monitored in
HL-60 cells. However, standard firefly luciferase
is unstable or degrades rapidly and the Renilla
luciferase should be employed instead (pRL vectors,
- Due to the low transfection efficiency of HL-60
cells, transient transfection assays should include a
readily visualizable marker of transfection such as
cotransfection with an EGFP plasmid, or preferably
using a vector that expresses both your gene of interest
and EGFP from the same plasmid (pIRES2-EGFP,
F. Inhibition of Gene Expression Using
- If transient assays are to be performed, use 15 btg
of supercoiled plasmid per transfection. If stable transfectants
are to be isolated, linearize each plasmid with
a restriction endonuclease that cleaves the vector but
leaves your gene of interest, the selectable marker, and
any regulatory elements intact. Solve either preparation
in a small volume of PBS.
- Collect HL-60 cells by centrifugation and resuspend
at 1.0 × 106 in growth medium. Combine 400µl
of cells with DNA in a 0.4-cm-gap electroporation
cuvette. Allow to stand for 5 min at room temperature.
- Mix the cuvette by gentle tapping and insert into
a square-wave electroporation device. Electroporate
with three pulses of 1500 V each and 90µs duration.
- Allow the cells to recover for 5 min and then plate
into 10ml of growth medium and incubate at 37°C in
8% CO2 in a humidified atmosphere.
- For transient assays, recover the cells the next
day and perform CAT or luciferase assays by standard
protocols. For monitoring of fluorescence from EGFP
expression, prepare the cells as in steps 1 and 2 of
the immunofluorescence protocol, mount the cells,
and examine for EGFP expression using a FITC filter
- For stable transfectant recovery, wait 24h and
then add appropriate selective agent. Geneticin and
hygromycin B are effective at 500 and 150µg/ml,
respectively, in HL-60 cells.
- Allow for selection of stable transfectants by
growth for 7-10 days. Cells can either be maintained
at this stage as a pooled group of transfectants or individual
clones can be isolated by single-cell dilution
and expansion in 96-well plates.
- Successful transfection in pools or individual
clones should be monitored either by immunofluorescence
or by Western blotting using either an antibody
against the protein or an epitope tag if present.
- Synthetic oligonucleotides should be synthesized
as 15-mers using standard base chemistry. The exact
concentration at which an oligonucleotide effectively
disrupts translation or targets a given mRNA for
RNase-mediated decay is a function of intracel-lular
concentration, expression level of the target, and ability
of the oligonucleotide to hybridize, i.e., secondary
structure limitations. Therefore, several oligonucleotides
and a range of oligonucleotide concentrations
with a suitable scrambled control oligonucleotide need
to be empirically tested. Initial experiments with siRNA
indicate that effective gene knockdown by this
approach can be performed transiently, but long-term
expression of dsRNA may induce an interferon
response (Bridge et al., 2003) in HL-60 cells.
- Prepare solutions of 5mg/ml transferrin and
5mg/ml insulin in unsupplemented RPMI 1640
medium. Prepare serum-free growth medium by
supplementing RPMI 1640 with standard concentrations
of penicillin-streptomycin (100µg/ml) and
L-glutamine (200 mM).
IV. COMMENTS AND PITFALLS
- Collect HL-60 cells by centrifugation and wash
three times in serum-free growth medium. Count the
cells by hemocytometer chamber counting and plate
the cells at 1 × 105 cells/ml in serum-free growth
- Supplement the cells with 5µg/ml transferrin
and 5 µ/ml insulin. Add a series of dilutions of your
test and control oligonucleotides spanning a concentration
range from 100 nM to 100 µM.
- Allow uptake of the oligonucleotide to occur
over a period of 6h incubation at 37°C in an 8% CO2 in a humidified atmosphere.
- Restore FBS levels to 10% and allow overnight
- Monitor toxicity to the cells by trypan blue dye
exclusion assays. Determine the effect of your test and
control oligonucleotides on the expression of your
gene of interest by either immunofluorescence or
reverse transcriptase polymerase chain reaction
- Subsequent effects on differentiation induction
and specific marker appearance can then be determined
by following steps 1-4 and then adding the
specific chemical inducer.
- When thawing materials (antibodies, antigen, or
samples), mix thoroughly before dilution or processing.
Avoid multiple freeze-thaw cycles by aliquoting
small volumes of reagents into multiple microcentrifuge
tubes for storage.
- When using all-trans retinoic acid, avoid exposure
- Fluorescent-activated cell sorting quantitation is
not recommended when the differentiation inducer
causes cell clumping, as is the case with PMA and
- For some inducers that cause attachment,
trypsin-EDTA treatment may not remove all cells from
the surface of tissue culture dishes. Therefore, bacteriological
grade petri dishes are utilized for procedures
that require removal of the attached cells.
- Use multiple differentiation markers when
testing the effect of a new inducer.
- In preparing cells for immunological analysis,
use heat-inactivated serum to eliminate cell killing due
to active complement.
This work was supported by the U. S. Department
of Energy, Office of Health and Environmental
Research, under Contract W-31-109-ENG-38, and the
National Institutes of Health under Grant CA80826.
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