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 cells with 20 μmol/L Cc, CC@CHLNV, and blank
CC@CHLNV.
5. Treat the cells for 24 h in an incubator at 5% CO 2 and 37
C,
digest the cells with 1 mL of 0.25% trypsin for 7 min, and
transfer the cell suspension to a centrifuge tube and centrifuge
at 1000 Â g for 3 min.
6. Discard the supernatant, wash the cells with 2–3 times PBS
solution, discard the supernatant, and add 70% cold ethanol to
fix the cells at 4
C for 24 h.
7. Add propidium iodide (PI) and RNase to the fixed cells, measure the cell cycle on a flow cytometer at 488 nm after incubation at 37
C for 30 min (see Note 13).
3.3.3 Determination of
Cell Apoptosis of Lewis
Lung Cancer 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. Cultivate 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 are attached.
4. Treat cells with 20 μmol/L Cc, CC@CHLNV, and blank
CC@CHLNV.
5. Incubate the cells for 24 h in an incubator at 5% CO 2 and
37
C, digest the cells with 1 mL of 0.25% trypsin for 7 min,
and transfer the cell suspension to a centrifuge tube and centrifuge at 1000 Â g for 3 min.
6. Transfer the cell pellet to a centrifuge tube and add 195 μL
Annexin V-FITC binding solution to prepare a suspension (see
Note 14).
7. Add 5 μL of Annexin V-FITC staining solution to the EP tube,
incubate on ice for 10 min in the dark.
8. Centrifuge at 1000 Â g for 3 min, discard the supernatant, and
add Annexin V-FITC binding solution to prepare a cell
suspension.
9. Add 10 μL of PI staining solution, mix well into a cell suspension, incubate on ice for 10 min in the dark.
10. Detect the samples by flow cytometry (see Note 15).
Catanionic Hybrid Lipid Nanovesicles
63
37
C for 12 h overnight until they were attached.
4. Treat cells with 20 μmol/L Cc, CC@CHLNV, and blank
CC@CHLNV.
5. Treat the cells for 24 h in an incubator at 5% CO 2 and 37
C,
digest the cells with 1 mL of 0.25% trypsin for 7 min, and
transfer the cell suspension to a centrifuge tube and centrifuge
at 1000 Â g for 3 min.
6. Discard the supernatant, wash the cells with 2–3 times PBS
solution, discard the supernatant, and add 70% cold ethanol to
fix the cells at 4
C for 24 h.
7. Add propidium iodide (PI) and RNase to the fixed cells, measure the cell cycle on a flow cytometer at 488 nm after incubation at 37
C for 30 min (see Note 13).
3.3.3 Determination of
Cell Apoptosis of Lewis
Lung Cancer 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. Cultivate 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 are attached.
4. Treat cells with 20 μmol/L Cc, CC@CHLNV, and blank
CC@CHLNV.
5. Incubate the cells for 24 h in an incubator at 5% CO 2 and
37
C, digest the cells with 1 mL of 0.25% trypsin for 7 min,
and transfer the cell suspension to a centrifuge tube and centrifuge at 1000 Â g for 3 min.
6. Transfer the cell pellet to a centrifuge tube and add 195 μL
Annexin V-FITC binding solution to prepare a suspension (see
Note 14).
7. Add 5 μL of Annexin V-FITC staining solution to the EP tube,
incubate on ice for 10 min in the dark.
8. Centrifuge at 1000 Â g for 3 min, discard the supernatant, and
add Annexin V-FITC binding solution to prepare a cell
suspension.
9. Add 10 μL of PI staining solution, mix well into a cell suspension, incubate on ice for 10 min in the dark.
10. Detect the samples by flow cytometry (see Note 15).
Catanionic Hybrid Lipid Nanovesicles
63