Hi, I'm Samaya Mad from Research Center for Pharmaceutical and Technology. Today we would like to show you how to uset assay to evaluate the availability of cells after 13 them with different plexes. Let's get started.
First, I remove the medium. Then I'm going to watch the cells with PBS. But first I change the people to avoid the contamination PBS.
Now with the new peoples, I wish the cells, I mix it gently and I remove the PBS completely. I use a smaller PPA two. I add a few microliters of trips.
Team, make sure that all the cells are covered with treeing. Now I'm tapping the flask. It helps to de attach the cells completely.
Now here you can see the cells are detached in inward microscope. I add medium to stop the tripsin. Having peeing, I want to make sure that all the cells are detached.
And now I transfer cells to Falcon two. Again by pee petting, I want to make sure that I have a homogeneous cell suspension. I transfer about 500 microliters of cell suspension into append of tube.
Then I take about 50 microliters of cell suspension to another micro tube and I add the same volume of trip on blue and I mix it To count the cells, I need a hemo meter first. I put a few drops of water here and here, and I gently place one cover glass onto this slide. I mix the sample completely just before counting and with the sample details, I put seven microliters of cell suspension into the counting chamber.
Be careful not to overload. I pull down The cell suspension in the trial reservoir and I used a channel transfer paper to see the cells in 96. Well play, I mix the cells to make sure that cells are suspended ly.
Then I continue to see the cells first, I remove the Medium, I changed the teeth, and I replace it with serum and antibody free medium. These cells will be incubated for 15 minutes at two seven degrees Celsius. To prepare the reaction media.
First I add film and antibody free media to all the, and then I add antisense and I mix it by preparing. I also add antisense to other samples. After adding antisense, I meet the sample completely and I add scramble.
Antisense sends to this sample And I mix it. Now I add different polymers to the samples and I mix them. Now I'm adding media.
First I remove serum free medium and I add the treatment to eat well. Again, for the other samples, I'm adding media. I mix them.
I removed serum free medium and I add the treatment to each well. And this is the last sample. I remove serum free medium and I add treatment to the last valve as after four hours, I remove the treatment media and I replenish cells with fresh media.
After 24 hours of incubation, I will perform entity assay. So I need 20 milliliters of DA without ol red, and I add four milliliters of filtered entity solution. Having petting, I would like to make sure that they were mixed well, and now I transfer it to a reserver.
Now I removed the media and I replace it with 250 microliters of MTT added demand. Up to here, all the wells have received empty except the four wells, which will fail as blank controls. For Solubilizing, the water insoluble for Amazon produced by metabolic active cells, I need to have 20 milliliters of DMSO plus 2.5 milliliters of so and sun's buffer.
I add DMSO and having appropriate petting, I want to make sure that they were mixed well. Now gently, I remove the TT added demand and I replace it with D initial plus instance buffer. And I put the plate in plate three there, and I can read the results with preset essay.
No matter what the treatment is, entity is a risk of method, so the viability of cells can be evaluated after any different treatment. N entity metabolism is proportional to the level of metabolism energy in the cells. Therefore, even in the absence of cell proliferation, metabolically activated cells can be evaluated.
With this method, very small number of cells can be detected. The form one production is proportional to the amount of entity in the incubation medium, and the maximum amount of offor on produced is not the same in different cell lines. The absorption of chromosome varies with the pH.
That's why most researchers at buffer at pH 10.5 in the case that you need more than one plate, make sure that controls are included in all the plates. This metal suffers from some minor disadvantages. First of all, metabolically inactivated cells cannot be discriminated with death cells.
MD solution is sensitive to light, so you should protect it from light, and it could be stored for a maximum of one month at four degrees Celsius. The cells that used for N-C-T-S-A cannot be further used for any other purposes and about the web, about the stability of drug. This method fail to validate the stability of drug in the medium, and that's all.
And thanks for your attention.
