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NEOPLASIA
From the Department of Medicine, Harvard Medical
School, and the Department of Adult Oncology and Department of
Biostatistical Science, Dana-Farber Cancer Institute, Boston, MA; and
the Breast Center, Baylor College of Medicine, Houston, TX.
Follicular lymphomas (FLs) are neoplastic counterparts of normal
germinal center (GC) B cells. FLs are characterized by t(14;18) with
deregulation of the Bcl-2 (BCL2) gene. The
presence of t(14;18) and overexpression of Bcl-2 is
necessary, but not sufficient, to cause this disease. An array
containing 588 complementary DNAs (cDNAs) was used to compare the gene
expression between GC B cells and FL cells. To specifically monitor
genes expressed in normal GC B and FL cells and not the entire tissue
compartment, normal and malignant B cells were purified from tissues.
Using the array, 37 genes were up-regulated and 28 were down-regulated
in FL cells as compared to normal GC B cells. The expression level of
each differentially expressed gene was verified by quantitative
polymerase chain reaction. Following these studies 24 genes were
up-regulated and 8 genes down-regulated with a P value less
than .1. Included among the genes that were up-regulated in FLs were
cell cycle regulator proteins CDK10, p120, p21CIP1, and p16INK4A;
transcription factors/regulators Pax-5 and Id-2, which are involved in
normal B-cell development; and genes involved in cell-cell
interactions, tumor necrosis factor, interleukin-2R Follicular lymphomas (FLs) are characterized by the
t(14;18) (q32;q21) translocation in virtually all patients. This
translocation involves the Bcl-2 gene (BCL2) on
chromosome 18 juxtaposed with the immunoglobulin heavy chain
(IGHG1) joining region on chromosome 14 (Bcl-2/IgH).1 This leads to deregulation of the
BCL2 gene, with overproduction of the antiapoptotic Bcl-2
protein. A substantial body of evidence supports the hypothesis that
the Bcl-2/IgH translocation is necessary, but not sufficient, to cause
FL. Compelling evidence comes from studies of transgenic mice that bear
a Bcl-2/IgH fusion gene to mimic the t(14;18) translocation in humans.
These mice developed polyclonal B-cell hyperplasia in lymph nodes and
spleen with IgM/IgD+ mature B cells, but not a monoclonal
lymphoproliferative disorder.2,3 Similar to the
progression seen in human FL, a minor subset of these transgenic mice
go on to develop a monoclonal diffuse large B-cell lymphoma that is
associated with a rearranged c-Myc (MYC) gene in about 50%
of cases.3
Further evidence that the Bcl-2/IgH translocation alone is not
causative for follicular non-Hodgkin lymphoma (NHL) is that B cells
with t(14;18) are present in a substantial number of healthy individuals. In peripheral blood, t(14;18)+ cells have been
reported in 8% to 88% of healthy subjects using polymerase chain
reaction (PCR). Similarly using fluorescein in situ hybridization
(FISH), B cells with t(14;18) have been detected in hyperplastic
lymphoid tissues (tonsil, lymph nodes) of 12% to 54% of healthy
individuals.4-6 Similar to the risk of FL increasing with
age, the detection of t(14;18) in peripheral blood of healthy humans
appears to increase with age.7 Cells with
t(14;18)+ have also been detected by PCR in peripheral
blood of patients with localized FL who are in long-term remission
(> 10 years) after radiation therapy.8 One
interpretation of these finding is that these cells lack other genetic
changes necessary for transformation into recurrent lymphoma.
One approach to understand neoplastic transformation is by comparing
gene expression of normal cells to their malignant counterparts. The
development of complementary DNA (cDNA) array technology allows one to
simultaneously quantify the expression of a very large number of
genes.9-18 The application of DNA array technology to studies of human malignancies includes identifying the gene profile of
malignancies of different cell types or lineages; genes involved in
pathogenesis, progression, and metastasis; genetically determined risk
groups; genes involved in resistance; and ultimately potential molecular targets of therapy. To date, gene profiling studies of NHL
have identified clinically relevant subgroups, related histologic
subgroups to normal cellular counterparts, and molecules that are
specific to certain disease entities.10,13 In the present
report, we have used a cDNA microarray to compare the expression of 588 genes in FL cells with their normal cellular counterparts, germinal
center (GC) B cells isolated from hyperplastic tonsils. The levels of
differentially expressed genes were verified by real-time quantitative
reverse transcription PCR (RT-PCR). Our data suggest that there is
differential expression of a large number of genes between normal GC B
cells and FL cells. Among these are transcription factors, cell cycle
regulators, signal transduction molecules, and molecules involved in
cell-cell interactions. These studies may provide further understanding
of the pathogenesis and pathophysiology of FL.
Cells
Flow cytofluorometric analysis and antibodies
Poly(A)+ RNAs extraction and cDNA labeling Cells were resuspended in TRIzol reagent (Gibco BRL, Grand Island, NY) according to the manufacturer's procedures. Contaminating DNA was removed by treatment with RNAse-free DNase I (Boehringer Mannheim, Indianapolis, IN) as described in the manufacturer's manual (Clontech, Palo Alto, CA). After 1 hour of incubation, the samples were extracted with phenol/chloroform/isoamylalcohol (25:24:1) and chloroform/isoamyl-alcohol (24:1). Total RNAs were finally precipitated with 1:5 (v/v) 7.5 M ammonium acetate and 1:2.5 (v/v) 100% ethanol. Poly(A)+ RNA was purified using Oligotex messenger RNA (mRNA) isolation kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. The cDNA probes were synthesized either from 1 µg mRNA extracted from normal GC B cells or FL cells by reverse transcription according to the Atlas cDNA Expression Array manufacturer's protocol (Clontech) using 35 µCi (1.295 KBq) [32P]dATP (3.000 Ci/mmol; 111 TBq/mmol)
(NEN, Boston, MA).
Probe purification and hybridization Reverse transcribed products were purified using CHROMA SPIN-200 DEPC-H2O columns as described by the manufacturer (Clontech). Incorporation of 32P into the probes was determined after counting an aliquot in a liquid scintillation counter (Packard, Meridien, CT). The fractions corresponding to the peak were pooled and denatured (Clontech). The Atlas cDNA Expression Array membranes containing 588 cDNAs (Clontech) were prehybridized in ExpressHyb buffer (Clontech) containing 100 µg/mL heat-denatured salmon sperm (Sigma, St Louis, MO) for 3 hours in a rotating oven at 68°C. The denatured probes were added directly into the hybridization buffer and further incubated for 18 hours at 68°C. The membranes were washed 4 times 30 minutes at 68°C with 2 × standard sodium citrate (SSC)-1% sodium dodecyl sulfate (SDS), twice 30 minutes at 68°C with 0.1 × SSC-0.5% SDS, and finally at room temperature for 5 minutes in 2 × SSC. Hybridizations were performed in duplicate. Duplicate measurements were analyzed for reproducibility. Quantitation was assessed and transformed by the logarithm (base 10). The ratios of the measurements had a median of 0.036; 17% of the measurements were either above 0.10 or below 0.10 on the log scale, suggesting that
83% of the genes could be replicated with no more than 10% error.
Image acquisition and DNA array analysis The membranes were exposed to phosphorimager screens at room temperature for 2 days (Molecular Dynamics, Sunnyvale, CA). The screens were scanned on a Storm 860 Phosphorimager (Molecular Dynamics) at a resolution of 50 µm. Scans were imported and arrayed using the AtlasImage 1.5 software (Clontech). The arrays from the 6 different normal GC B-cell samples were averaged after normalization of their intensities using the sum method as described in the AtlasImage 1.5 user manual. The same procedure was applied to the arrays from the 6 different FL samples. Using this methodology -actin (GenBank
accession no. X00351, Human Genome Organization (HUGO) nomenclature
ACTB) and ribosomal protein S9 (GenBank accession no. U14971,
HUGO nomenclature RPS9) were equally expressed in all the samples. Two
composite arrays were generated, respectively. These 2 composite arrays
were finally compared using the AtlasImage 1.5 software. Intensity of
the genes ranged from 0 to 65 280 arbitrary units (AU). For a given
gene, positive ratio corresponded to the normalized value of the
intensity of a gene in FL cells divided by the normalized value in
normal GC B cells. Negative ratios are the negative value of the ratio
of the normalized gene intensities of normal GC B cells divided by the
one of FL cells. Ratios greater than or equal to 2 and less than or
equal to 2 were identified for further investigation. To include
genes for which the value was below background level (ie, 0 for the
AtlasImage 1.5 software), we took into consideration differences in
intensities (FL minus GC) greater than or equal to 2000 and less than
or equal to 2000 AU. All the expressed genes that did not meet these
criteria are available at
http://dfciwww.dfci.harvard.edu/AO/Freedman_Lab/cDNA_array/not_different_expressed.html.
Real-time quantitative PCR analysis Real-time quantitative RT-PCR analysis was performed using an ABI PRISM 7700 Sequence Detection System instrument using the SYBR Green I dye (PE Biosystems, Foster City, CA). Briefly, direct detection of PCR product was monitored by measuring the increase in fluorescence caused by the binding of SYBR Green I dye to double-stranded DNA by the Sequence Detection System directly into the reaction tube. The sequence detector software 1.7 calculates the threshold cycle number (CT) when signals reach 10 times the SD of the baseline. It was previously demonstrated that the calculated CT values are a quantitative measurement for the mRNA levels of various genes tested.20,21 DNase I-treated total RNA (1 µg) was reverse transcribed using the Advantage RT-for-PCR kit (Clontech) following the manufacturer's instructions, resuspended in 100 µL final volume, and aliquoted to avoid freezing and thawing procedures. RT reaction (2.5 µL), or water as control, was amplified in triplicate by real-time PCR in a final volume of 50 µL using the SYBR Green Master Mix reagent at a final concentration of 1 × (PE Biosystems). The appropriate primers were designed with the Primer Express 1.0 software (PE Biosystems). To ensure that any DNA contamination was removed by DNAse I treatment, real-time RT-PCR was performed on non-reverse-transcribed RNA. No amplification was observed in these conditions for all 65 genes that were differentially expressed. Due to the nonspecific binding of SYBR Green I to double-stranded DNA, the optimal primer concentrations were defined after verifying that no amplification was observed in the no template controls (NTCs) for a given set of forward and reverse primer concentrations. To ensure the specificity of the reaction, the size of the PCR product for each gene was verified by gel electrophoresis. The sequences and the concentrations of the primers are available at http://dfciwww.dfci.harvard.edu/AO/Freedman_Lab/cDNA_array/sequences.html. Conditions were as follows: 1 cycle at 50°C for 2 minutes, 1 cycle at 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. Validation experiments to verify the efficiencies of amplification of the primers of target and reference genes were approximately equal.For each sample, CT values for Statistical analysis Genes were identified from the Clontech array as described above. Results of quantitative RT-PCR were assessed by the 2-sample t test; nominal P values are presented without correction for multiple comparisons.
Cell surface analysis of purified GC B cells and FL cells To try to better understand the differences in gene expression between normal GC B cells and FL cells we used purified cell populations. Single mononuclear cell suspensions of tonsil and tissues biopsies involved with FL contain significant numbers of T cells, NK cells, and monocytes and in the case of tonsil, naive and memory B cells. To limit the interference of any genes from the microenvironment in our study, we used immunomagnetic bead depletion to remove these cells from our preparations. Normal GC B cells were purified from single-cell suspensions of tonsils. The immunophenotype of GC B cells has been previously described to be CD20+/CD38+ and IgD /CD44.19,22 Following
immunomagnetic bead depletion of T cells, monocytes, NK cells, and
IgD+/CD44+ cells, the population of cells were
phenotyped and analyzed by flow cytometry. The preparation of GC B
cells was more than 98% CD20+/CD38+, with no
detectable CD4+, CD8+, CD56+,
CD14+, CD44+, and IgD+ cells.
Similarly, immunomagnetic depletion of T cells, NK cells, and monocytes
was performed with single-cell suspensions of tissues involved with FL.
Cell surface marker analysis of FL cells showed a monoclonal population
CD20+ B cells.23-25
cDNA array analysis In an attempt to further understand differences between FL and normal GC B cells, we compared the gene expression between these 2 cell types by cDNA microarray. In the Clontech array the randomly selected cDNA represented all known sequences spotted onto a nylon membrane as amplified products of 200 to 600 base pairs (bp) in length. These fragments have been carefully selected from gene regions that lack the poly-A tail, repetitive elements, or other highly homologous sequences. The array is organized in 6 different quadrants each containing one or several gene families. The families include oncogenes, tumor suppressors, cell cycle control proteins (Figure 1A); ion channels and transport, intracellular signal transduction modulators and effectors, stress response proteins (Figure 1B); apoptosis, DNA synthesis, repair and recombination proteins (Figure 1C); transcription factors, general DNA-binding proteins (Figure 1D); receptors, cell surface antigens, cell adhesion (Figure 1E); and cell-cell communication factors (Figure 1F).
Radiolabeled probes for hybridization were derived from 2 sources of
mRNA: GC B cells derived from the tonsils of 6 healthy individuals and
FL cells isolated from tissues of 6 patients with relapsed disease. Six
arrays obtained after hybridization of the 6 GC B-cell probes were
averaged as well as the 6 arrays from 6 FL probes to create 2 composite
arrays, respectively. Finally, after global normalization and
subtraction of the background, the 2 composite arrays were compared.
The expression of actin and S9 were constant in all 12 samples
analyzed. Figure 1 summarizes the genes for which the ratio of FL to GC
was more than 2 or less than Real-time quantitative RT-PCR analysis Although differences in gene expression between FL and normal GC B cells were observed with the cDNA array, it was critical to verify these results using another methodology. We monitored the mRNA level of all genes noted to be differentially expressed in the Atlas array, according to the criteria described above by real-time quantitative RT-PCR using SYBR Green 1.20,26 We also monitored Bcl-2, which is increased in the vast majority of FL as compared to normal GC B cells27 as well as 2 housekeeping genes (-actin and
ribosomal protein S9). Results representative of real-time quantitative
PCR are shown Figure 2 for Bcl-2,
-actin, and ribosomal protein S9. The CT value at which
the fluorescence reaches the threshold is higher for GC than for FL
cells, which is reflective of a lower mRNA content for Bcl-2 in GC B
cells. The CT values obtained for GC or FL for -actin as
well as ribosomal S9 protein are the same, indicating that the mRNA
content was identical in these 2 samples. The NTCs remained below the
threshold, showing that no amplification was observed without
template.
Genes increased in FL Besides the increase of Bcl-2 in FL, relatively few genes have been shown to be increased as compared to normal B cells. Among these genes are those for Pax-5 (PAX5), TNF (TNF) and CXCR4 (SDF-1 receptor).28-30 To confirm the differential expression of the 37 genes that were increased in FL cells, we performed real-time quantitative RT-PCR analysis. The CT values were normalized for each gene in all of the samples obtained from the GC B and FL cells as described in "Materials and methods." The data were analyzed using a 2-sample t test. As seen in Table 1, 24 genes are listed, 21 that were found to be increased in FL using the array (P < .1), and 3 genes whose expression was decreased in FL by the array. The difference between the mean of the CT value for FL and the mean of the CT value for GC was calculated ( mean
CT). These 24 genes are listed in descending order of their
level of expression ( mean CT). As reported previously,
approximately 2.5 CT values correspond to a 10-fold
increase.26 Consistent with prior reports, we found that
Bcl-2, TNF, and Pax-5 were all overexpressed in FL
cells.1,13,28,29 In FL cells, several genes encoding for
cell cycle regulator proteins were overexpressed including CDK10, p120
(NOL1), p21CIP1 (CDKN1A), and p16INK4A
(CDK2A). Considering that FL cells are often in
G0/G1, it is of interest that regulators of
G1 phase p21CIP1 and p16INK4A were overexpressed. A large
number of transcription factors and DNA binding proteins were
preferentially expressed in FL cells including Bsp-1
(MADH1); Id-2 (ID2); c-Jun (JUN);
v-erbA (ERBAL2); ZFX (ZFX); Db1
(ZNF161); Pax-5; SNF2- (SMARCA2); and Hsf-1
(HSF1). There is evidence that Id-2 and Pax-5 are both
important in B-cell differentiation. A set of genes that are involved
in microenvironmental interactions including cytokines and cytokines
receptors were also overexpressed in FL cells including TNF, IL-4R,
IL-2R. Three genes (YY1, Lyn, cyclin B1
[CCNB1]), which had decreased expression in FL cells using
the cDNA array, were overexpressed by quantitative RT-PCR with P
values of less than .02, .08, and .03, respectively.
Genes decreased in FL There is relatively little information on genes that are down-regulated in FL cells, as compared to normal B cells. We confirmed by RT-PCR the decreased expression in 7 of the 28 genes noted to be down-regulated in FL cells using the array (P < .1), whereas in 21 genes the decrease noted using the array was not confirmed by RT-PCR. One gene (LFA1), which was increased in FL by array, was found to be decreased by RT-PCR. Table 2 shows these 8 genes in descending order of their level of expression in GC B cells ( mean
CT). The majority of genes are involved in
microenvironmental or cell-cell interactions including
MRP14; MRP8; CD40; thymosin 10 (TMSB10); and DBI (DBI). Consistent with the low
proliferative index of FL cells, the cell cycle regulator p55CDC
(CDC20) had decreased expression in the FL cells.
Validation sample We conducted a small validation study of the 32 genes that were significantly different in their expression levels. Three additional FL samples were studied by RT-PCR only. Of the 24 genes found to be overexpressed in FL cells by RT-PCR, 15 were confirmed to be overexpressed in FL cells compared to GC cells in this small validation sample. Of the 8 genes that had decreased expression in FL cells, 5 were confirmed to be significantly decreased in the 3 additional cases. An asterisk in Tables 1 and 2 indicates these genes.
Follicular lymphomas have been hypothesized to be neoplastic transformations of normal GC-B cells. This was supported first by morphology, and then by virtue of the expression of various cell surface antigens.31,32 However the finding that FL cells possess in essentially all cases the Bcl-2/IgH rearrangement and overexpression of Bcl-2 protein provided clear distinction from normal GC B cells. In the present report we have compared the gene expression profile of FL cells to normal tonsillar GC B cells. Based on the evidence that Bcl-2/IgH rearrangement and overexpression of Bcl-2 is necessary but not sufficient to induce FL, we hypothesized that additional alterations in gene expression may contribute to the malignant phenotype. Our objective was to identify genes that were differentially expressed in the malignant cells and their normal cellular counterparts using a cDNA array and confirm expression using real-time quantitative RT-PCR as a second platform. For a limited number of genes the results of quantitative PCR differed from the array data. This was unexpected, but emphasizes the need to confirm array data using a second methodology. We identified a series of genes that were either overexpressed or underexpressed in FL cells as compared to normal GC B cells. A number of these genes are known to be important in normal and malignant B cells and may help explain certain aspects of the pathophysiology of FL. Because all patients had relapsed disease, our data may not apply to FL cells isolated from untreated patients. Furthermore, it is possible that some of the alterations in gene expression were related to prior cytotoxic therapy. The localization of certain of these genes at sites of known chromosomal abnormalities in FL may provide further evidence for their role in the malignant transformation. The genes that had the greatest Several transcription factors/regulators and DNA binding proteins were
overexpressed in FL cells. Among these were Pax-5 and Id-2, both of
which have important roles in B-cell development and
differentiation.29,41-44 Pax-5, also known as
B-cell-specific activator protein (BSAP), is a DNA binding protein.
Pax-5 expression is restricted to B cells, the testis in adults, and
the brain and liver in the fetus. In B-cell ontogeny, Pax-5 mRNA is
expressed from the pro-B-cell stage through mature B cells, decreasing
in plasma cells. Mice lacking Pax-5 have B-cell maturation arrest at
the pre-B-cell stage. Pax-5 regulates expression of several B-cell-specific genes including CD19, the tyrosine kinase Blk, and
pre-B-cell-specific surrogate light chain genes.45 Pax-5 localizes to 9p13 and has deregulated expression in lymphoplasmacytic lymphomas and some cases of diffuse large cell lymphoma with
t(9;14).46,47 Our studies are consistent with a
previous report that the majority of cases of FL overexpress Pax-5
protein.29 It is unclear what role Pax-5 plays in FL;
however, overexpression in splenic B cells and cell lines enhances
proliferation and modulates p53 expression.48 Similar to
Pax-5, Id-2 has been shown to be important in B-cell development. Id
proteins are helix-loop-helix transcription regulators that play an
important role in hematopoiesis.49 Id proteins bind to
other helix-loop-helix-bearing transcription factors, inhibiting their
function.50 In mice, Id-2 is expressed in pro-B cells and
decreases during differentiation.51 In normal peripheral blood mononuclear cells, Id-2 mRNA levels decrease with mitogen stimulation.50 Id-2 plays a critical role in lymphoid
tissue development because Id-2 The FL cells reside in a GC microenvironment within lymphoid tissues
and in the marrow in association with other lymphohematopoietic and
stromal cells.53 Evidence indicates that interactions of FL cells with components of the microenvironment modulate the growth
and survival of the malignant cells.54 FL cells had
increased mRNA expression of 2 cytokine receptors: IL-2R One problem with comparing gene expression of normal and neoplastic
tissues is the difficulty of determining whether the signal is
originating from the tumor cells or from associated normal cells.
Within normal tonsils and tissue involved with FL, there are many
normal cellular constituents (ie, fibroblasts, monocytes, T, NK,
endothelial cells, and FDCs). Although the microenvironment in FL may
in fact differ from that of normal lymphoid tissues, in this study we
only sought to examine gene expression in the B cells. The GC B cells
used in this study were representative of a total population, rather
than separated into centroblasts or centrocytes. The phenotype of the
GC B cells used was CD38+, CD44 Follicular lymphomas have been compared to normal GC B cells using
oligonucleotide microarrays.13 Seventeen of the 32 genes we confirmed by RT-PCR to be differentially expressed were present on
the microarray used by Alizadeh et al.13 Despite a
different technical approach, a number of the genes that we found to be overexpressed in FL cells were also increased in the array analysis by
Alizadeh et al. These include BCL2, BSP1, JUN, IL4R,
IL-2R In addition to the t(14;18) translocation, additional cytogenetic
abnormalities have been described in FL.66-72 Additional chromosomal abnormalities involving, for example, p53, play a central
role in the progression of FL. These additional abnormalities are
likely to be important in the development of the disease, because
Bcl-2/IgH is necessary but not sufficient for tumor development. These
changes include gains and losses of chromosomal material that can
either lead to an increased or a decreased expression of other genes.
In this study we found a number of genes that were deregulated and
localized to sites of known genetic abnormalities observed in FL (Table
3). For example, p21CIP1 and TNF,
assigned to 6p21.1 and 6p21.3, respectively, are located within
high-level DNA amplifications on band 6p21.71
MRP8, MRP14, and TDPX2 genes are
located within commonly seen chromosome 1 breakpoints 1q21-22 and
1p22-36, respectively.66,69
In conclusion, we have identified a number of genes that are differentially expressed in FL cells and their normal counterparts, GC B cells, using a combination of gene array technology and quantitative PCR analysis. These studies focused on only the normal and neoplastic B cells by using highly purified cells without contaminating cells from the associated microenvironment. Several of the genes identified are involved in regulating the cell cycle, and their overexpression in FL cells is consistent with their low proliferative state. The observation that a number of transcription factors overexpressed by FL cells, particularly Pax-5 and Id-2, have a central role in normal B-cell development, may lead to better understanding of the regulation of other genes involved in the pathogenesis and pathophysiology of this disease. In addition to insights into the biology of FL, comparing gene expression in normal B cells and FL cells may lead to the identification of potential targets as well as antigens for immunotherapeutic strategies.
Submitted October 10, 2000; accepted August 23, 2001.
Supported in part by National Institutes of Health grants CA55207 and CA66996, and the Norman Hirschfield Foundation. H.H. was supported by the Cure for Lymphoma Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Arnold S. Freedman, Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 02115; e-mail: arnold_freedman{at}dfci.harvard.edu.
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Top
Abstract
Introduction
or 
light
chains (Southern Biotechnology Associates, Birmingham, AL). An
irrelevant isotype-matched control mAb was used in all experiments. The
secondary antibody was a phycoerythrin R-conjugated goat F(ab')2
antimouse IgG (Southern Biotechnology Associates) diluted at 2 µg/mL.
Flow cytometry was carried out within 2 hours using an EPICS XL
(Beckman-Coulter, Hialeah, FL). Cells were gated using forward versus
side scatter to exclude dead cells and debris.
