the dish after which the donor cells are added. Thus, we recommend seeding the acceptor cells earlier than the donor cells.
A typical time gap between the plating of the acceptor cells and
the donor cells is about 3.5–4 h.
6. It is essential to collect as many cells as possible after trypsinization. Thus, we recommend incubating with trypsin for about
5 min in the incubator, with occasional shaking, to detach as
many cells as possible. It is recommended to use an inverted
light microscope to determine if cells have been effectively
detached. The trypsinization time can vary with cell types.
7. To visualize the transfer of individual mRNA in co-culture of
adherent cells, one can employ single-molecule FISH using
fluorescent mRNA probes specific for the gene of interest, as
described in [27]. Using Cy3-labeled probes specific for the
MS2 loops (comprising 35 end-labeled 5
0 and 3
0 amino-allyl
oligonucleotides [21]), we found that about 0.7% (~7 copies
per cell) of β-actin-MBS mRNA transfers from MBS-MEF to
MCF7 during 3 h. However, by 12 h, about 2% of β-actin-MBS
mRNA (~30 copies per cell) underwent transfer (Fig. 4).
8. After allowing the cell suspension to pass through the magnetic
column, the negatively selected fraction is collected in the
eluate. To collect the positively selected fraction of high purity,
it is essential to press the plunger through the column immediately after adding media to the column. If the media is allowed
to passively elute through the column, the efficiency of recovery of the positively selected cells (MCF7) may decrease.
9. The amount of RNA collected can vary between cell types, cell
numbers, and kits used. For reference, we use the Qiagen
RNeasy mini kit, which allowed us to extract up to 32 μg of
total RNA from 3 x 10
6 MBS-MEF cells and 38 μg of total
RNA from 3 Â 10
6 MCF7 cells. When using Macherey-Nagel
NucleoSpin
® RNA, we could collect 49 μg and 50 μg of total
RNA from same number of MBS-MEF and MCF7 cells,
respectively.
10. The presence of any nonspecific band is detected using
DNA-PCR, such as the human MALAT1 band in the MBSMEF-enriched fraction post-sorting, would indicate that sorting was not sufficiently efficient and the resulting human and
mouse cell fractions are not pure. Under this situation, we
recommend passing magnetic microbead-labeled cell suspension through a new LS column for one or more times, as
described in Subheading 3.2.
11. For the verification of RNA transfer using RT-PCR, we recommend to use a high amount of input RNA for the first-strand
cDNA synthesis (1–5 μg) to be able to detect the less abundantly transferred RNAs by semiquantitative PCR. If no PCR
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Sandipan Dasgupta and Jeffrey E. Gerst
A typical time gap between the plating of the acceptor cells and
the donor cells is about 3.5–4 h.
6. It is essential to collect as many cells as possible after trypsinization. Thus, we recommend incubating with trypsin for about
5 min in the incubator, with occasional shaking, to detach as
many cells as possible. It is recommended to use an inverted
light microscope to determine if cells have been effectively
detached. The trypsinization time can vary with cell types.
7. To visualize the transfer of individual mRNA in co-culture of
adherent cells, one can employ single-molecule FISH using
fluorescent mRNA probes specific for the gene of interest, as
described in [27]. Using Cy3-labeled probes specific for the
MS2 loops (comprising 35 end-labeled 5
0 and 3
0 amino-allyl
oligonucleotides [21]), we found that about 0.7% (~7 copies
per cell) of β-actin-MBS mRNA transfers from MBS-MEF to
MCF7 during 3 h. However, by 12 h, about 2% of β-actin-MBS
mRNA (~30 copies per cell) underwent transfer (Fig. 4).
8. After allowing the cell suspension to pass through the magnetic
column, the negatively selected fraction is collected in the
eluate. To collect the positively selected fraction of high purity,
it is essential to press the plunger through the column immediately after adding media to the column. If the media is allowed
to passively elute through the column, the efficiency of recovery of the positively selected cells (MCF7) may decrease.
9. The amount of RNA collected can vary between cell types, cell
numbers, and kits used. For reference, we use the Qiagen
RNeasy mini kit, which allowed us to extract up to 32 μg of
total RNA from 3 x 10
6 MBS-MEF cells and 38 μg of total
RNA from 3 Â 10
6 MCF7 cells. When using Macherey-Nagel
NucleoSpin
® RNA, we could collect 49 μg and 50 μg of total
RNA from same number of MBS-MEF and MCF7 cells,
respectively.
10. The presence of any nonspecific band is detected using
DNA-PCR, such as the human MALAT1 band in the MBSMEF-enriched fraction post-sorting, would indicate that sorting was not sufficiently efficient and the resulting human and
mouse cell fractions are not pure. Under this situation, we
recommend passing magnetic microbead-labeled cell suspension through a new LS column for one or more times, as
described in Subheading 3.2.
11. For the verification of RNA transfer using RT-PCR, we recommend to use a high amount of input RNA for the first-strand
cDNA synthesis (1–5 μg) to be able to detect the less abundantly transferred RNAs by semiquantitative PCR. If no PCR
212
Sandipan Dasgupta and Jeffrey E. Gerst