The isolations of genuine populations of apoptotic reversal cells has been a problem previously, which greatly limits our understanding on the consequences of apoptosis reversal. This protocol allow us to generate a purer populations of apoptotic reversal cells. The main advantage of this technique is that the materials and equipment used are widely available in different institution.
This technique is readily applicable for isolating a purer populations of cells that have undergone reversal of apoptosis. As previously reported, normal cells can also go undergo apoptosis reversal, that apart from breast cancer cells, different chemo cells, as well as normal cells, can be started and the various apoptotic stimuli can be used. Because the reversal cell have gone through apoptosis and fast sorting, the recovery rate is usually low.
A larger starting cell number is recombinant to increase the final yield. To begin, culture MCF-7, MDA-MB-231, To begin, culture MCF-7, MDA-MB-231, and T47D cells, according to the text protocol. After this, wash the cells with PBS twice, then add two milliliters of 0.05 percent trypsin-EDTA of 0.05 percent trypsin-EDTA to the MCF-7, and MDA-MB-231 cells, to the MCF-7, and MDA-MB-231 cells, and two milliliters of 0.25 percent trypsin-EDTA and two milliliters of 0.25 percent trypsin-EDTA to the T47D cells.
to the T47D cells. Culture the cells for five minutes at 37 degrees Celsius, in a five percent carbon-dioxide cell culture incubator. While the cells incubate, check the detachment of the cells with a microscope to prevent over-digestion by trypsin-EDTA.
When more than 90 percent of the cells detach, add five milliliters of completed RPMI medium to the dish, and pipette it over the cell layer surface several times. Then, transfer the cells to 15 milliliter conical tubes, and centrifuge. Discard the supernatant, and re-suspend the pellets with FACS buffer in 1.5 milliliter micro-centrifuge tubes.
Then, divide the cells into several tubes. Centrifuge the tubes once more, and discard the supernatant. Then, add diluted Fc block to the samples.
Incubate the samples on ice in the dark for 20 minutes, before centrifuging the samples again. After this, discard the supernatant. Next, add fluorochrome-conjugated monoclonal antibodies against human CD44, and CD24, against human CD44, and CD24, in one to 40, and one to 10 dilutions, respectively, to the dual-staining groups.
For the positive controls, add CD44 antibodies For the positive controls, add CD44 antibodies to the MDA-MB-231 cells, to the MDA-MB-231 cells, and CD24 antibodies to the MCF-7 cells and CD24 antibodies to the MCF-7 cells in one to 40, and one to 10 dilutions, respectively. Add PerCP-Cy5.5 Mouse IgG2b Kappa, Add PerCP-Cy5.5 Mouse IgG2b Kappa, diluted at a one to 40 ratio to cells as an isotope control for CD44 antibodies. Then, add PE Mouse IgG2a Kappa, Then, add PE Mouse IgG2a Kappa, diluted at a one to 10 ratio to the cells as an isotope control for CD24 antibodies.
Incubate the samples at four degrees Celsius in the dark for 30 minutes. Then, centrifuge the samples at 300 g's Then, centrifuge the samples at 300 g's and four degrees Celsius for five minutes. Discard the supernatant, and wash the pellet twice in 500 micro-liters of PBS.
Then, repeat the centrifugation as previously described. Re-suspend the pellet on 0.5 milliliters of PBS, and filter the suspension through a 40 micrometer nylon mesh. Then, run the suspension through a fluorescence-activated cell sorter.
Collect the cells with CD44 negative, and CD24 positive markers in round-bottom tubes containing one milliliter of collection medium. Then, centrifuge the tubes, and discard the supernatant. Next, plate the sorted breast non-stem cancer cells in a culture dish containing fresh collection medium for further culture.
First, use one millimolar staurosporine in DMSO, To prepare 2.5 micro-molars staurosporine in medium, according to the text protocol. Then, remove the culture medium from the cells. Wash the cells with two milliliters of PBS once, after this, add 10 milliliters of the staurosporine medium to the MSF-7 cells for six hours, to induce apoptosis, when the cells are 70 percent confluent.
Next, prepare one millimolar paclitaxel in DMSO. Then, add 12.5 micro-liters Then, add 12.5 micro-liters of the 1 millimolar paclitaxel to the completed medium of the T47D cells to make up a final volume of 10 milliliter in a 15 milliliter conical tube. Remove the culture medium from the cells, and wash the cells with 2 milliliters of PBS.
Then, add the paclitaxel medium to the cells for 10 hours to induce apoptosis for 10 hours to induce apoptosis when the cells reach 70 percent confluency. After this, remove the medium from the solvent-treated MCF-7 group, and wash them with two milliliters of PBS. Then, add 10 milliliters of 0.25 percent DMSO medium Then, add 10 milliliters of 0.25 percent DMSO medium for six hours as the solvent control for staurosporine.
Remove the culture medium from the solvent-treated T47D group, and wash the cells with two milliliters of PBS. Then, add 10 milliliters of 0.05 percent DMSO medium Then, add 10 milliliters of 0.05 percent DMSO medium for 10 hours as the solvent control for paclitaxel. Next, stain the cell and incubate them for 20 minutes at 37 degrees Celsius in a five percent carbon-dioxide incubator.
Use a 60 times confocal laser-scanning microscope to observe the morphological changes of the treated cells. Stain both the apoptotic inducer and solvent-treated MCF-7 and T47D cells and solvent-treated MCF-7 and T47D cells in the dark for 30 minutes at 37 degrees Celsius in a five percent carbon-dioxide incubator. Using the fluorescence-activated cell sorter, collect the positive cells from the inducer-treated groups in round bottom tubes containing one milliliter of collection medium.
Then, centrifuge the tubes as previously described, and discard the supernatant. Collect the negative cells from the solvent-treated groups in round-bottom tubes with one milliliter of collection medium. Centrifuge the tubes, and discard the supernatant.
After this, re-suspend the sorted cells in fresh collection medium. Seed the cells in 12-well tissue culture plates, and culture them for seven days for apoptosis reversal. Harvest the reversed MCF-7 in both inducer and solvent-treated groups with 0.05 percent trypsin-EDTA.
with 0.05 percent trypsin-EDTA. Then, harvest the T47D cells in both inducer Then, harvest the T47D cells in both inducer and solvent-treated groups with 0.25 percent trypsin-EDTA. Stain the cells with fluorochrome-conjugated monoclonal antibodies against human CD44 and CD24.
While the cells stain, prepare the isotype controls as previously demonstrated. Finally, run the cells on a flow cytometer, and detect the percentage of cells with CD44 positive, and CD24 negative markers. In this protocol, the transition from breast non-stem cancer cells to breast CSC-like cells was observed.
Isolation of non-stem cancer cells was first done in MCF-7 cells. Typical morphological changes were observed after adding apoptotic inducers, and the cells recovered from apoptosis with similar morphology after drug withdrawal. The caspace-activated cells were labeled and sorted out based on their higher-fluorescense intensity than cells without caspace activation.
During the apoptotic induction process, solvent was used to exclude the possibility that the facts procedure, or the solvent itself, was the cause for the transition. Caspace-activated cells in the apoptotic-inducer treatment groups were found to be a nexin five positive and PI negative, suggesting that they were apoptotic cells. The apoptotic cells were collected and cultured for recovery, compared with the solvent-treated groups, flow cytometric analysis showed that there were cells appearing in the CD44 positive and CD24 negative quadrant in the reversed originally breast non-stem cancer cell population.
Gaging is important in flow analysis and cell sorting, that sufficient control should be prepared in the labs. Since this in vitro apoptosis reversal procedure isolates pure apoptotic cancer cells, experiments such as in vivo tumorigenesis assets can be conducted to understand the consequences of real reverse the cells. This technique sheds light on the cause for cancer relapse, therefore, it can be investigated to potentially prevent the re-occurrence in cancer patients.
Please remember to protect yourself properly when conducting cell culture experiments according to the biosafe-tier regulations at your institution.
