3.5 Flow Cytometry
1. At the end of each EMT or MET phases, recover cells to be
analysed by flow cytometry.
2. Stain freshly harvested cells with Live/Dead cell viability solution for 20 min in obscurity. For E-cadherin staining, block
cells in 5% FBS/1% BSA/PBS for 30 min and incubate with
PE-conjugated anti-E-cadherin (1:10 dilution in 5% FBS/1%
BSA/PBS).
3. Fix cells using a solution of 1% PFA/PBS for 30 min, permeabilize with 0.1% Triton-X100/PBS for 20 min and block with
5% FBS/1% BSA/PBS for 30 min.
4. Following PBS washes, incubate cells with APC-conjugated
anti-Fibronectin (1:10 dilution in 5% FBS/1% BSA/PBS) for
20 min at 4
C. All incubations and staining are done at 4
C.
5. Analyse stained cells by flow cytometry to determine
E-cadherin and Fibronectin expression levels (Fig. 3a and
Note 7). Analyse data with FlowJo software (Tree Star,
Ashland, OR).
Fig. 3 (a) E-Cadherin and Fibronectin levels were quantified by flow cytometry. Mean Fluorescence Intensity
(MFI) is shown for each half EMT/MET cycle for control cells (blue circles, not treated) and EMT/MET cycling
cells (red square). (b) SNAIL1, SNAIL2 and ZEB1 expression levels quantified by real-time PCR. Relative
expression levels are shown for each half EMT/MET cycle for control cells (blue circles, not treated) and
EMT/MET cycling cells (red square). C: cycles
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1. At the end of each EMT or MET phases, recover cells to be
analysed by flow cytometry.
2. Stain freshly harvested cells with Live/Dead cell viability solution for 20 min in obscurity. For E-cadherin staining, block
cells in 5% FBS/1% BSA/PBS for 30 min and incubate with
PE-conjugated anti-E-cadherin (1:10 dilution in 5% FBS/1%
BSA/PBS).
3. Fix cells using a solution of 1% PFA/PBS for 30 min, permeabilize with 0.1% Triton-X100/PBS for 20 min and block with
5% FBS/1% BSA/PBS for 30 min.
4. Following PBS washes, incubate cells with APC-conjugated
anti-Fibronectin (1:10 dilution in 5% FBS/1% BSA/PBS) for
20 min at 4
C. All incubations and staining are done at 4
C.
5. Analyse stained cells by flow cytometry to determine
E-cadherin and Fibronectin expression levels (Fig. 3a and
Note 7). Analyse data with FlowJo software (Tree Star,
Ashland, OR).
Fig. 3 (a) E-Cadherin and Fibronectin levels were quantified by flow cytometry. Mean Fluorescence Intensity
(MFI) is shown for each half EMT/MET cycle for control cells (blue circles, not treated) and EMT/MET cycling
cells (red square). (b) SNAIL1, SNAIL2 and ZEB1 expression levels quantified by real-time PCR. Relative
expression levels are shown for each half EMT/MET cycle for control cells (blue circles, not treated) and
EMT/MET cycling cells (red square). C: cycles
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Cecile Davaine et al.