19. LS Columns (Miltenyi Biotec).
20. LD Columns (Miltenyi Biotec).
21. QuadroMACS separator or suitable magnetic separator.
3 Methods
3.1 Isolation of EMT
Populations from YFPExpressing Tumors
by FACS
1. Dissolve collagenase IV in DMEM/F12 at 2 mg/mL and
prewarm at 37
C in a water bath.
2. Remove the tumor-bearing tissue (i.e., pancreas or implantable
tumor). Be careful to only remove the tumor portion and not
contaminating normal tissue. Transfer to a 6-well dish and
wash tissue three times with cold 1Â PBS. After the third
wash, leave a small amount of liquid to enhance cutting of the
tissue.
3. Mince the tissue into small pieces with dissection scissors (alternatively a razor blade and a Petri dish lid can be used) (see Note
3).
4. Using a transfer pipette, take a small amount of the 2 mg/mL
pre-warmed collagenase and transfer the tissue into 10 mL of
collagenase in a 15 mL conical tube.
5. Incubate the tissue at 37
C in a water bath in dark for 20 min.
Vortex or shake vigorously every 5 min (see Note 4).
6. Transfer the now digested tumor mixture into a 50 mL conical
tube using a 70 μm cell strainer. Using the plunger of a 3 mL
syringe, push the tissue through the filter. Pour several mLs of
cold DMEM/F12 and continue to push tissue until large
pieces are dissociated.
7. Remove and discard the cell strainer and fill the tube to 50 mL
with ice cold DMEM/F12 (see Note 5).
8. Centrifuge solution at 300 RCF for 5 min at 4
C.
9. Carefully remove and discard the supernatant, being careful
not to disturb or aspirate the mobile cell pellet. Add 500 μL
ice cold-staining buffer and transfer cells to a microfuge tube.
10. Centrifuge cells at 4
C at 300 RCF for 5 min at 4
C and
discard the supernatant.
11. Resuspend cells in 520 μL ice cold-staining buffer and save a
small aliquot of cells (~20 μL) as an unstained negative control
(this will receive secondary antibody only in the following steps
(see Note 6)). In the remaining 500 μL solution, add 2 μL
(1:250) rat anti-ECAD primary antibody and incubate on ice
for 15 min.
Isolation and Identification of EMT Subtypes
319
20. LD Columns (Miltenyi Biotec).
21. QuadroMACS separator or suitable magnetic separator.
3 Methods
3.1 Isolation of EMT
Populations from YFPExpressing Tumors
by FACS
1. Dissolve collagenase IV in DMEM/F12 at 2 mg/mL and
prewarm at 37
C in a water bath.
2. Remove the tumor-bearing tissue (i.e., pancreas or implantable
tumor). Be careful to only remove the tumor portion and not
contaminating normal tissue. Transfer to a 6-well dish and
wash tissue three times with cold 1Â PBS. After the third
wash, leave a small amount of liquid to enhance cutting of the
tissue.
3. Mince the tissue into small pieces with dissection scissors (alternatively a razor blade and a Petri dish lid can be used) (see Note
3).
4. Using a transfer pipette, take a small amount of the 2 mg/mL
pre-warmed collagenase and transfer the tissue into 10 mL of
collagenase in a 15 mL conical tube.
5. Incubate the tissue at 37
C in a water bath in dark for 20 min.
Vortex or shake vigorously every 5 min (see Note 4).
6. Transfer the now digested tumor mixture into a 50 mL conical
tube using a 70 μm cell strainer. Using the plunger of a 3 mL
syringe, push the tissue through the filter. Pour several mLs of
cold DMEM/F12 and continue to push tissue until large
pieces are dissociated.
7. Remove and discard the cell strainer and fill the tube to 50 mL
with ice cold DMEM/F12 (see Note 5).
8. Centrifuge solution at 300 RCF for 5 min at 4
C.
9. Carefully remove and discard the supernatant, being careful
not to disturb or aspirate the mobile cell pellet. Add 500 μL
ice cold-staining buffer and transfer cells to a microfuge tube.
10. Centrifuge cells at 4
C at 300 RCF for 5 min at 4
C and
discard the supernatant.
11. Resuspend cells in 520 μL ice cold-staining buffer and save a
small aliquot of cells (~20 μL) as an unstained negative control
(this will receive secondary antibody only in the following steps
(see Note 6)). In the remaining 500 μL solution, add 2 μL
(1:250) rat anti-ECAD primary antibody and incubate on ice
for 15 min.
Isolation and Identification of EMT Subtypes
319