3.3.4 Determination of
Intracellular Active Oxygen
Concentration by Flow
Cytometry
1. Under the microscope, select the logarithmic growth phase
cells, wash twice with sterile PBS solution, digest with 2 mL
of 0.25% trypsin for 7 min, centrifuge at 1000 Â g for 5 min,
collect the cells, and add 4 mL of complete medium to prepare
a cell suspension.
2. Cultivating Lewis lung cancer cells at a concentration of
3–5 Â 10
5 cells in each well and inoculate them into 6-well
plates.
3. Culture the inoculated cells in an incubator at 5% CO 2 and
37
C for 12 h overnight until they were attached.
4. Treat the cells with 20 μmol/L CC, CC@CHLNV, and blank
CC@CHLNV and culture the cells in an incubator at 5% CO 2
and 37
C for 24 h, remove the complete medium, add DCFHDA dye (10 μmol/L) to the solution and incubate for 20 min
in the incubator at 5% CO 2 , 37
C (see Note 14).
5. Wash the cells three times with medium containing no serum.
6. Measure the change in fluorescence intensity of the cell sample
by flow cytometry. Set the excitation wavelength to 488 nm
and the emission wavelength to 525 nm.
3.3.5 Determination of
the Intracellular Calcium
Ion Concentration by Flow
Cytometry
1. Under the microscope, select the logarithmic growth phase
cells, wash twice with sterile PBS solution, digest with 2 mL
of 0.25% trypsin for 7 min, centrifuge at 1000 Â g for 5 min,
collect the cells, and add 4 mL of complete medium to prepare
a cell suspension.
2. Cultivating Lewis lung cancer cells at a concentration of
3–5 Â 10
5 cells in each well and inoculate them into 6-well
plates.
3. Culture the inoculated cells in an incubator at 5% CO 2 and
37
C for 12 h overnight until they were attached.
The operation steps are the same as those in Subheading
3.3.2, steps 1–3.
4. Treat the cells with 20 μmol/L CC, CC@CHLNV, and blank
CC@CHLNV and culture the cells in an incubator at 5% CO 2
and 37
C for 24 h, remove the medium, digest the cells by
adding 2 mL of 0.25% trypsin for 7 min to cell suspension.
5. Centrifuge at 1000 Â g for 3 min, discard the supernatant,
wash the cells three times with PBS.
6. Add Fluo-3 fluorescent probe to the cell suspension and allow
staining for 30 min (see Note 14).
7. Measure the change in fluorescence intensity of the cell sample
by flow cytometry. Set the excitation wavelength to 488 nm
and the emission wavelength to 525 nm.
64
Xuemei Xie et al.
Intracellular Active Oxygen
Concentration by Flow
Cytometry
1. Under the microscope, select the logarithmic growth phase
cells, wash twice with sterile PBS solution, digest with 2 mL
of 0.25% trypsin for 7 min, centrifuge at 1000 Â g for 5 min,
collect the cells, and add 4 mL of complete medium to prepare
a cell suspension.
2. Cultivating Lewis lung cancer cells at a concentration of
3–5 Â 10
5 cells in each well and inoculate them into 6-well
plates.
3. Culture the inoculated cells in an incubator at 5% CO 2 and
37
C for 12 h overnight until they were attached.
4. Treat the cells with 20 μmol/L CC, CC@CHLNV, and blank
CC@CHLNV and culture the cells in an incubator at 5% CO 2
and 37
C for 24 h, remove the complete medium, add DCFHDA dye (10 μmol/L) to the solution and incubate for 20 min
in the incubator at 5% CO 2 , 37
C (see Note 14).
5. Wash the cells three times with medium containing no serum.
6. Measure the change in fluorescence intensity of the cell sample
by flow cytometry. Set the excitation wavelength to 488 nm
and the emission wavelength to 525 nm.
3.3.5 Determination of
the Intracellular Calcium
Ion Concentration by Flow
Cytometry
1. Under the microscope, select the logarithmic growth phase
cells, wash twice with sterile PBS solution, digest with 2 mL
of 0.25% trypsin for 7 min, centrifuge at 1000 Â g for 5 min,
collect the cells, and add 4 mL of complete medium to prepare
a cell suspension.
2. Cultivating Lewis lung cancer cells at a concentration of
3–5 Â 10
5 cells in each well and inoculate them into 6-well
plates.
3. Culture the inoculated cells in an incubator at 5% CO 2 and
37
C for 12 h overnight until they were attached.
The operation steps are the same as those in Subheading
3.3.2, steps 1–3.
4. Treat the cells with 20 μmol/L CC, CC@CHLNV, and blank
CC@CHLNV and culture the cells in an incubator at 5% CO 2
and 37
C for 24 h, remove the medium, digest the cells by
adding 2 mL of 0.25% trypsin for 7 min to cell suspension.
5. Centrifuge at 1000 Â g for 3 min, discard the supernatant,
wash the cells three times with PBS.
6. Add Fluo-3 fluorescent probe to the cell suspension and allow
staining for 30 min (see Note 14).
7. Measure the change in fluorescence intensity of the cell sample
by flow cytometry. Set the excitation wavelength to 488 nm
and the emission wavelength to 525 nm.
64
Xuemei Xie et al.