4. Harvest the co-culture by incubating with 3 mL trypsin-EDTA
solution at 37
C for 5 min followed by neutralization with
3–5 mL of complete medium.
5. Pass the cell suspension through a 40 μm cell strainer.
6. (Optional, but highly recommended) In order to check for the
efficiency of cell sorting, an aliquot of ~5–10 Â 10
4 cells of the
“co-culture” sample can be saved for analysis by flow cytometry.
7. Spin down the cells in a centrifuge at 350 Â g for 10 min at 4
C
and aspirate the media carefully.
8. Resuspend the cells in 400 μL of cold DMEM and proceed to
magnetic bead-mediated cell sorting.
3.2 Magnetic
Bead-Mediated Cell
Sorting
1. Add human CD326-conjugated magnetic microbeads to cell
suspensions prepared above (Subheadings 3.1.2 and 3.1.3). In
the current protocol, MCF7 cells display surface antigen
CD326 (EpCAM). By using microbeads conjugated to antiCD326 antibodies, the MCF7 cells can be selected for and the
MBS-MEF cells, which do not possess the CD326 surface
marker, selected against. Use the microbeads at a concentration
recommended by the manufacturer.
2. Incubate the cell suspension as per the manufacturer’s instructions. For anti-CD326 microbeads, the recommended duration is 30 min on ice.
3. After 30 min of incubation, attach the MidiMACS Separator to
the MACS MultiStand.
4. Insert the LS Column into the MidiMACS Separator and add
3 mL of cold complete media or the sorting buffer to the LS
column. Allow the media/buffer to elute through the column
into a tube placed underneath the separator.
5. Place a 15 mL tube under the separator to collect the eluate
(the negatively selected fraction of the cells, i.e., the MBS-MEF
cells in this case).
6. Add the cell suspension (after incubation with the microbeads)
to the separator and let it pass through the column.
7. Wash the column twice with 1 mL of ice-cold medium or
sorting buffer. Remove the tubes with the eluate and keep on
ice (see Note 4).
8. Place another 15 mL tube under the separator to collect the
positively selected fraction of cells from the suspension (i.e., the
MCF7 cells in this case).
9. Add 3 mL of cold medium/sorting buffer to the column.
Immediately recover the positive fraction with the help of a
plunger (as supplied with the column, see Note 8).
10. (Optional) In order to check for the efficiency of cell sorting,
an aliquot of ~5–10 Â 10
4 cells of the sorted cell populations
202
Sandipan Dasgupta and Jeffrey E. Gerst
solution at 37
C for 5 min followed by neutralization with
3–5 mL of complete medium.
5. Pass the cell suspension through a 40 μm cell strainer.
6. (Optional, but highly recommended) In order to check for the
efficiency of cell sorting, an aliquot of ~5–10 Â 10
4 cells of the
“co-culture” sample can be saved for analysis by flow cytometry.
7. Spin down the cells in a centrifuge at 350 Â g for 10 min at 4
C
and aspirate the media carefully.
8. Resuspend the cells in 400 μL of cold DMEM and proceed to
magnetic bead-mediated cell sorting.
3.2 Magnetic
Bead-Mediated Cell
Sorting
1. Add human CD326-conjugated magnetic microbeads to cell
suspensions prepared above (Subheadings 3.1.2 and 3.1.3). In
the current protocol, MCF7 cells display surface antigen
CD326 (EpCAM). By using microbeads conjugated to antiCD326 antibodies, the MCF7 cells can be selected for and the
MBS-MEF cells, which do not possess the CD326 surface
marker, selected against. Use the microbeads at a concentration
recommended by the manufacturer.
2. Incubate the cell suspension as per the manufacturer’s instructions. For anti-CD326 microbeads, the recommended duration is 30 min on ice.
3. After 30 min of incubation, attach the MidiMACS Separator to
the MACS MultiStand.
4. Insert the LS Column into the MidiMACS Separator and add
3 mL of cold complete media or the sorting buffer to the LS
column. Allow the media/buffer to elute through the column
into a tube placed underneath the separator.
5. Place a 15 mL tube under the separator to collect the eluate
(the negatively selected fraction of the cells, i.e., the MBS-MEF
cells in this case).
6. Add the cell suspension (after incubation with the microbeads)
to the separator and let it pass through the column.
7. Wash the column twice with 1 mL of ice-cold medium or
sorting buffer. Remove the tubes with the eluate and keep on
ice (see Note 4).
8. Place another 15 mL tube under the separator to collect the
positively selected fraction of cells from the suspension (i.e., the
MCF7 cells in this case).
9. Add 3 mL of cold medium/sorting buffer to the column.
Immediately recover the positive fraction with the help of a
plunger (as supplied with the column, see Note 8).
10. (Optional) In order to check for the efficiency of cell sorting,
an aliquot of ~5–10 Â 10
4 cells of the sorted cell populations
202
Sandipan Dasgupta and Jeffrey E. Gerst