Supplementary data for “Dlk1 in Normal and Abnormal Hematopoiesis” by
Sakajiri S. et al.
Real-time RT-PCR assay
Total RNA was isolated from cell lines and clinical samples using Trizol reagent
(Gibco, BRL) according to the standard protocol. Total RNA (1g) was processed
directly to cDNA by reverse transcription with Superscript Ⅱ (Life Technologies, Inc.),
according to the manufacture’s protocol in a total volume of 20 l.
The primer pairs used for Dlk1 were 5’-AAG GAC TGC CAG AAA AAG
GAC-3’(forward), 5’-GCA GAA ATT GCC TGA GAA GC-3’ (reverse). The probe for
the Dlk1 was 5’- GCC TCC CAT GCC TCC TGC CT-3’. The primer pairs used for
Integrin 3 were 5’-GCC TCT GGG CTC ACC TCG CTG -3’ (forward). The primer
pairs used for 18S were 5’- AAA CGG CTA CCA CAT CCA AG-3’ (forward), 5’-CCT
CCA ATG GAT CCT CGT TA-3’ (reverse). The probe for the 18S was 5’-AGC AGG
CGC GCA AAT TAC CC-3’. Primers were purchased from Life Technologies, Inc., and
probes were from Perkin-Elmer Applied Biosystems.
For Dlk1 and 18S genes, amplification reactions contained 5 l of cDNA, 12.5 l of
the Universal Taqman 2x PCR master mix (Applied Biosystems), and 2.5 l of each of
the specific primers and probe. Primer and TaqMan probe concentrations in the final
volume of 25 l were 500 nM and 100 nM, respectively. All reactions were performed
in triplicate in an iCycler iQTM system (Biorad, Hercules, CA), and the thermal
cycling conditions were as follows: 2 min at 50C, 10 min at 95C, followed by 45
cycles of 95C for 15 sec and 60C for 1 min. For Integrin 3, the cDNAs were
subjected to PCR with SYBR GreenPCR Core Reagents (PE Applied Biosystems,
Foster City, CA) according to the manufacture’s protocol.
Levels of mRNA for each gene were evaluated as a ratio to the level of ribosomal
18S RNA to standardize the quantity of cDNAs of each sample. Furthermore, levels of
Dlk1 mRNA from the various samples were quantified as relative values to those
present in K562 cells; the relative value of Dlk1 transcripts in K562 cells as measured
by Real-time RT-PCR, was regarded as 100.
Colony formation assays.
For colony forming assays, we used 6-8 weeks old mice. Peripheral blood was
obtained by heart puncture and bone marrow was flushed from isolated femurs with
Alpha Minimum Essential Medium (-MEM) (Gibco BRL, Grand Island, New York,
USA) including 10% FBS using a 26-gauge needle. Isolated spleens were injected with
DMEM (Gibco BRL) plus 10% FCS, and crushed with forceps to release the cells.
Mononuclear cells from both bone marrow and spleens were separated by Ficoll
Hypaque density centrifugation (Amersham Pharmacia, Uppsala, Sweden).
Resuspended mononuclear bone marrow cells and growth factors were added 1:10
to methylcellulose medium M3234 (Stem Cell Technologies Inc., Vancouver, Canada)
to yield a final concentration of 1% methylcellulose, 30% FBS, 1% BSA, 10-4 M
mercaptoethanol, and 2 mM L-glutamine. Colony formation was stimulated by multiple
combinations of purified recombinant growth factors at the following final
concentrations: murine GM-CSF at 10 ng/mL; murine G-CSF at 10 ng/mL; murine IL-3
at 10 ng/mL; human IL-7 at 10 ng/mL; and human Epo at 2 U /mL. mDlk1 protein was
purified as previously described (16). Cells were cultured in six-well plates in a volume
of 1 ml and incubated at 37C in a humidified atmosphere containing 5% CO2. Colonies
were counted at various times. When using IL-3 and GM-CSF, colonies were
enumerated after 8 days culture. When using either G-CSF or IL-7, colonies were
counted after 12 days. Colonies (>50 cells) were enumerated with an inverted
microscope. Colony type was established by morphology, and to ensure accurate
determination, representative colonies were plucked from the plates, centrifuged onto
slides, stained with Wright-Giemsa stain and examined by light microscopy. Myeloid
and erythroid colony assays were performed by plating 2104 and 2105 bone marrow
cells, respectively; CFU-E colonies (consisting of >8 cells) were counted after 3 days
culture with EPO, and BFU-E colonies (consisting of >100 cells) were counted after 7
days culture with Epo and IL-3. The colony type was established by morphology; and to
ensure accuracy, BFU-E colonies were stained with Benzidine. For the megakaryocyte
colony-forming unit (CFU-Meg) assay, bone marrow cells (1105) were cultured with
MegaCult-C media (StemCell Technologies, Vancouver, Canada) in the presence of 10
ng/ml recombinant murine IL-3 (Calbiochem, Darmstadt, Germany), 20 ng/ml
recombinant human IL-6 (Calbiochem), and 50 ng/ml recombinant human TPO
(Calbiochem). After culturing for 7 days, colonies were stained with acetylcholine
esterase (AchE). CFU-Meg colonies were defined as at least 3 megakaryocytes in a
cluster.