patient with late stage pancreatic cancer. You are given the task of identifying
ID: 255746 • Letter: P
Question
patient with late stage pancreatic cancer. You are given the task of identifying pancreatic cancer stem cells in order to determine their sensitivity to the chemotherapeutic agent paclitaxel. Previous studies suggest that pancreatic cancer stem cells express the cell surface markers CD44 and CD24 isolate the cancer stem cell population. For each experiment, describe how you will process the sample and illustrate the results that will indicate that you have isolated pancreatic cancer stem cells Write your answers in the respective spaces below Expt 1Explanation / Answer
Experiment 1
Isolation of pancreatic stem cells from human xenografts
Protocol
1.First the specimen is collected from patient undergoing the treatment for pancreatic cancer.This is transplanted into mice and the tumor xenograft is collected.
2. The the tumour is put into cell dissociation buffer.The sample is weighed and then cut into pieces.These pieces are then transfered back into dissociation buffer and vortexed.This suspension is incubated for 2 hours vortexing every 20 mins.
3.Now after incubation filter through 70 micron filter and then centrifuge using ficoll paque plus.We should not mix ficoll with our filtrate.Collect the viable cells and stain with tryphan blue and count the number of viable cells in haemocytometer.If there are any dead cells or debris then again centriguge by density gradient centrifugation.
4. label some small tubes as unstained (1), then CD44(2) and CD 24(3), aldeflour reagent(4) and aldeflor+cd44+cd24(5).Aldeflour reagent is used to isolate cells showing positive aldehyde dehydrogenase activity.Dilute our cell suspension to final concentration of 5-10 millions cell/ml and keep on ice. Then add 100 Microliter to 1,2,3.Then in remaining cell suspension add aldeflour reagent and then tranfer into 5 and incubate for 40min.centriguge and then to pellets add aldeflour buffer and antibodies for cd44 and cd 24.
5. CD44+,CD24+ cells are collected based on gates created using cells stained with ALDEFLUOR, antibodies using the FL-4 and FL-2 channels.
Results
Cell showing positive aldehyde dehydrogenase activity will be collected and using antobodies of CD44 and CD 24 CSC's will be isolated.
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