3.3.6 Determination of
Mitochondrial Membrane
Potential 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 medium, digest the cells by
adding 2 mL of 0.25% trypsin for 7 min to cell suspension.
5. Add Rhodamine123 fluorescent probe to the cell suspension,
let stain for 30 min (see Note 14).
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.
7. Stain the tumor cells with Rhodamine, Fluo 3, and DCFH for
0.5 h and determine the fluorescence intensity using flow
cytometry FACS Vantage (see Note 18).
3.4 Antitumor
Efficiency In Vivo
1. Randomly divide tumor-bearing mice into four groups. Intraperitoneally give 0.9% NaCl, blank CC@CHLNV, CC or
CC@CHLNV using a 1 mL syringe to each group. Intraperitoneally administer with 50 μg CC once every 48 h for each
animal. After continuous administration for 14 days, observe
the body condition of the mice every day (see Note 19), record
the weight, and measure the size of the transplanted tumor
with a vernier caliper.
2. Cut the external skin along the root of the tumor with surgical
scissors, peel off the tumor tissue slowly from the skin using
ophthalmic tweezers and surgical scissors. Obtain tumor tissue
and record its weight. Sacrifice the mice by cervical dislocation.
Isolate the tumor tissue with a surgical scissor, weight tumor
tissue with a ten thousandth balance. (see Note 20).
4 Notes
1. Glycerin monostearate is used as solid lipid, and isopropyl
palmitate is used as liquid lipid substance.
2. Lipoid S 75 is used as zwitter-ionic surfactant and emulsifying
agent.
Catanionic Hybrid Lipid Nanovesicles
65
Mitochondrial Membrane
Potential 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 medium, digest the cells by
adding 2 mL of 0.25% trypsin for 7 min to cell suspension.
5. Add Rhodamine123 fluorescent probe to the cell suspension,
let stain for 30 min (see Note 14).
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.
7. Stain the tumor cells with Rhodamine, Fluo 3, and DCFH for
0.5 h and determine the fluorescence intensity using flow
cytometry FACS Vantage (see Note 18).
3.4 Antitumor
Efficiency In Vivo
1. Randomly divide tumor-bearing mice into four groups. Intraperitoneally give 0.9% NaCl, blank CC@CHLNV, CC or
CC@CHLNV using a 1 mL syringe to each group. Intraperitoneally administer with 50 μg CC once every 48 h for each
animal. After continuous administration for 14 days, observe
the body condition of the mice every day (see Note 19), record
the weight, and measure the size of the transplanted tumor
with a vernier caliper.
2. Cut the external skin along the root of the tumor with surgical
scissors, peel off the tumor tissue slowly from the skin using
ophthalmic tweezers and surgical scissors. Obtain tumor tissue
and record its weight. Sacrifice the mice by cervical dislocation.
Isolate the tumor tissue with a surgical scissor, weight tumor
tissue with a ten thousandth balance. (see Note 20).
4 Notes
1. Glycerin monostearate is used as solid lipid, and isopropyl
palmitate is used as liquid lipid substance.
2. Lipoid S 75 is used as zwitter-ionic surfactant and emulsifying
agent.
Catanionic Hybrid Lipid Nanovesicles
65