214
4. Measure out the rest of the volume (100 mL) into 150 mL
beaker.
5. Add DMEM powder slowly to the 1000 mL beaker while stirring gently. Stir until it is dissolved.
6. Rinse the original DMEM package with a small amount of
pure water from the 150 mL beaker.
7. Add this to the solution and any remaining pure water in step 4.
8. Add 3.7 g of sodium bicarbonate to the solution.
9. Stir with mechanical homogenizer until it is dissolved (see
Note 7).
10. While stirring, adjust pH of solution using 1 N HCl and 1 N
NaOH until it reaches the desired pH (7.4).
11. Sterilize the prepared solution (DMEM) by sterile filtering
using a bottle-top filter (in 0.22 μm pore size) into 2500 mL
sterile bottles in the hood.
12. Label the container with the name of media, sterility, and date
of preparation.
13. Store the prepared media in the refrigerator.
To evaluate the anticancer activity effect of ceranib-2 in polymeric
nanoform, the in vitro MTT assay was performed using SW480
and Hela cancer cell line incubated with PREC-2 NPs for 24 h.
DMSO was used as a carrier in the negative control (CNT), and
etoposide (50 μM) was used as the positive control. The results are
presented in Tables 2 and 3, and Fig. 10.
1. Seed Dukes’ type B colorectal adenocarcinoma (SW480) and
cervix adenocarcinoma (HeLa) cells in a 96-well flat-bottom
microtiter plate at a density of 1 × 10
4
cells/well in 100 μL
DMEM supplemented with 10% FBS (see Note 8).
2. Treat cells with various concentrations of the PREC-2 for 24 h
at 37 °C in a CO 2 incubator and 95% relative humidity (see
Notes 9 and 10).
3. After 24 h of incubation, replace with a fresh medium.
4. Add 10 μL of MTT working solution (5 mg/mL in phosphate
buffer solution) to each well and incubate the plate for 4 h at
37 °C in a CO 2 incubator (see Note 11).
5. Aspirate the medium, and solubilize the formed formazan
crystals by adding 50 μL of DMSO per well for 30 min at
37 °C in a CO 2 incubator (see Note 12).
6. Finally, quantify the intensity of the dissolved formazan crystals (purple color) using the ELISA plate reader at 540 nm (see
Note 13).
7. Schematic presentation of assay format is given in Fig. 10.
3.7 Determination
of the Efficacy
and Therapeutic Effect
of PREC-2 NPs
Ali Ben Taleb et al.
4. Measure out the rest of the volume (100 mL) into 150 mL
beaker.
5. Add DMEM powder slowly to the 1000 mL beaker while stirring gently. Stir until it is dissolved.
6. Rinse the original DMEM package with a small amount of
pure water from the 150 mL beaker.
7. Add this to the solution and any remaining pure water in step 4.
8. Add 3.7 g of sodium bicarbonate to the solution.
9. Stir with mechanical homogenizer until it is dissolved (see
Note 7).
10. While stirring, adjust pH of solution using 1 N HCl and 1 N
NaOH until it reaches the desired pH (7.4).
11. Sterilize the prepared solution (DMEM) by sterile filtering
using a bottle-top filter (in 0.22 μm pore size) into 2500 mL
sterile bottles in the hood.
12. Label the container with the name of media, sterility, and date
of preparation.
13. Store the prepared media in the refrigerator.
To evaluate the anticancer activity effect of ceranib-2 in polymeric
nanoform, the in vitro MTT assay was performed using SW480
and Hela cancer cell line incubated with PREC-2 NPs for 24 h.
DMSO was used as a carrier in the negative control (CNT), and
etoposide (50 μM) was used as the positive control. The results are
presented in Tables 2 and 3, and Fig. 10.
1. Seed Dukes’ type B colorectal adenocarcinoma (SW480) and
cervix adenocarcinoma (HeLa) cells in a 96-well flat-bottom
microtiter plate at a density of 1 × 10
4
cells/well in 100 μL
DMEM supplemented with 10% FBS (see Note 8).
2. Treat cells with various concentrations of the PREC-2 for 24 h
at 37 °C in a CO 2 incubator and 95% relative humidity (see
Notes 9 and 10).
3. After 24 h of incubation, replace with a fresh medium.
4. Add 10 μL of MTT working solution (5 mg/mL in phosphate
buffer solution) to each well and incubate the plate for 4 h at
37 °C in a CO 2 incubator (see Note 11).
5. Aspirate the medium, and solubilize the formed formazan
crystals by adding 50 μL of DMSO per well for 30 min at
37 °C in a CO 2 incubator (see Note 12).
6. Finally, quantify the intensity of the dissolved formazan crystals (purple color) using the ELISA plate reader at 540 nm (see
Note 13).
7. Schematic presentation of assay format is given in Fig. 10.
3.7 Determination
of the Efficacy
and Therapeutic Effect
of PREC-2 NPs
Ali Ben Taleb et al.