2. Human breast carcinoma (MCF7) cells:
Although the protocol does not depend on using tagged
RNAs, we strongly recommend to check the efficiency of the sorting protocol by verifying the transfer of a bona fide transferred
mRNA. In this protocol, β-actin-MBS mRNA is used as a model
transferred mRNA from MBS-MEF (donor) to MCF7 (acceptor)
cells.
It is important to note that the protocol can be adapted for use
with other pairs of heterologous adherent mammalian cells, as well
as with other co-culture models (see Notes 1 and 2). In addition,
the protocol can be scaled up to sort a higher number of cells, as per
the intended design of the experiment. If available, use of a
pre-validated transferred mRNA is highly recommended to determine the efficiency of transfer.
2.1 Cell Culture
1. Dulbecco’s modified Eagle medium (DMEM) high-glucose
culture medium.
2. Composite penicillin-streptomycin antibiotic solution:
10,000 units/mL penicillin G, 10 mg/mL streptomycin.
3. 11 mg/mL (100 mM) Sodium pyruvate solution.
4. European-grade fetal bovine serum.
5. Trypsin-EDTA solution: 0.25% Trypsin, 0.02% EDTA.
6. 150 mm Sterile cell culture dishes.
7. 0.5–1 mg/mL Fibronectin from bovine plasma: Dilute the
stock solution of fibronectin to a working solution of 0.5 μM
in warm PBS.
8. 1Â Phosphate-buffered saline (PBS): 0.137 M NaCl,
0.0027 M KCl, 0.01 M Na 2 HPO 4 , 0.0018 M KH 2 PO 4 .
9. Hemocytometer or cell counter.
2.2 Magnetic Sorting
1. 15 mL Volume conical tubes.
2. 40 μm Nylon cell strainer.
3. Temperature-controlled centrifuge for spinning 15 mL volume
tubes.
4. MidiMACS Separator for separating up to 10
9 cells (Miltenyi
Biotec).
5. MACS MultiStand (Miltenyi Biotec).
6. LS Columns (Miltenyi Biotec).
7. Human CD326 (epithelial cell adhesion molecule; EpCAM)
magnetic microbeads (Miltenyi Biotech).
8. Alexa Fluor
® 488-labeled anti-human CD326 (EpCAM) antibody (Biolegend).
9. 7-Aminoactinomycin D (7-AAD) for monitoring cell viability.
198
Sandipan Dasgupta and Jeffrey E. Gerst
Although the protocol does not depend on using tagged
RNAs, we strongly recommend to check the efficiency of the sorting protocol by verifying the transfer of a bona fide transferred
mRNA. In this protocol, β-actin-MBS mRNA is used as a model
transferred mRNA from MBS-MEF (donor) to MCF7 (acceptor)
cells.
It is important to note that the protocol can be adapted for use
with other pairs of heterologous adherent mammalian cells, as well
as with other co-culture models (see Notes 1 and 2). In addition,
the protocol can be scaled up to sort a higher number of cells, as per
the intended design of the experiment. If available, use of a
pre-validated transferred mRNA is highly recommended to determine the efficiency of transfer.
2.1 Cell Culture
1. Dulbecco’s modified Eagle medium (DMEM) high-glucose
culture medium.
2. Composite penicillin-streptomycin antibiotic solution:
10,000 units/mL penicillin G, 10 mg/mL streptomycin.
3. 11 mg/mL (100 mM) Sodium pyruvate solution.
4. European-grade fetal bovine serum.
5. Trypsin-EDTA solution: 0.25% Trypsin, 0.02% EDTA.
6. 150 mm Sterile cell culture dishes.
7. 0.5–1 mg/mL Fibronectin from bovine plasma: Dilute the
stock solution of fibronectin to a working solution of 0.5 μM
in warm PBS.
8. 1Â Phosphate-buffered saline (PBS): 0.137 M NaCl,
0.0027 M KCl, 0.01 M Na 2 HPO 4 , 0.0018 M KH 2 PO 4 .
9. Hemocytometer or cell counter.
2.2 Magnetic Sorting
1. 15 mL Volume conical tubes.
2. 40 μm Nylon cell strainer.
3. Temperature-controlled centrifuge for spinning 15 mL volume
tubes.
4. MidiMACS Separator for separating up to 10
9 cells (Miltenyi
Biotec).
5. MACS MultiStand (Miltenyi Biotec).
6. LS Columns (Miltenyi Biotec).
7. Human CD326 (epithelial cell adhesion molecule; EpCAM)
magnetic microbeads (Miltenyi Biotech).
8. Alexa Fluor
® 488-labeled anti-human CD326 (EpCAM) antibody (Biolegend).
9. 7-Aminoactinomycin D (7-AAD) for monitoring cell viability.
198
Sandipan Dasgupta and Jeffrey E. Gerst