G6PD and Cancer

2 things to be done for this coursework : first , it is the abstract for a PhD thesis. Secondly, use the software : FlowJo or any other software to combine each experiment’s histograms that i will attach to you into 1 figure that combine all the histograms for each experiment in 1. In other words, 1 figure for 1 experiment, all I need is that the figures look like all other histograms that appear in the published papers for the same types of experiments so I can add them into my thesis and my paper later . Then , write explanations for the data, i mean analyze the figures and write explanations underneath them all.( no words limit). Also, explain the purpose and the results of all the rest of the experiments. Let me explain my research: the General name of the thesis is : G6PD Deficiency and Cancer. ( specific name of the abstract is NOT decided yet , the writer can decide it based on the information that is given to him here ) I received on 2017-10-11 tissue sample from 33 years patient with cervical cancer .. I had very difficult time at the beginning to grow and culture the primary cells in vitro as the tissue didn’t adhere quickly, and the cells grew so slow until I decided to sort the cells that are grown by using BD influx -activated cell sorter machine and selected fibroblasts cells . So I am working now on these fibroblasts Cervical cancer cells. I followed the methods and the protocol that are stated in the attached published paper . The researcher that published this paper worked in HeLa Cells ,so the difference between my research and his is the type of cells. Maybe I will add more methods to the methods he used such as : AFM and maybe more , but I still didn’t finish so I will think what to add more later to improve the research enable to publish a good paper. The research is consist of 2 parts. I use 100mm treated petri dish plate to grow my fibroblasts cervical cancer cells, then I move them to 6Wells plate before I start adding the medicine to perform each experiment. The medicine that I am using to treat the fibroblasts cells is : Nicotinamide I am also using 1 well in all the plates that I name it NAC : n- acetyl-l-cysteine.( the purpose is to wash/ clean the ROS) In part 1 of the research, the methods i used are( all are explained in the attached paper ) ; – CCk8 experiment. – DAPI experiment. To see the morphology. – Apoptosis experiment – ROS experiment – MDA experiment ( read the plate 3 times- attached). The formula is : Concentration of MDA( nmol/mg) = sample measurement OD – control OD/ standard OD- Blank OD X concentration of standard (10nmol/ ml) / concentration of sample protein ( mg/ml) – GSH experiment/ read the plates 3 times: ( there is something wrong in the analysis as the diagram for the 4mg/ml concentration comes with minus ( the opposite side ) after I did all the calculations by using the formula and subtract the blank wells but I still don’t know what is wrong with results and why it is coming with minus, so please revise the excel sheet maybe I calculated something wrong – i did the experiment 2 times) GSH formula nmol/mg= measure OD value- Blank OD well value /standard OD value- Blank OD value X 20nmol/ml / concentration of Protein mg/ml – JC-1 to measure the potential Mitochondria membrane. This what I have completed so far , I performed the Western Blot once but I forgot to add the cleaved antibodies so the results are not completed yet . So I still have to perform Western Blot again. I am using : Caspase 3 and cleaved Caspase 3 Caspase 8 and cleaved Caspase 8 Caspase 9 and cleaved Caspase 9 Internal antibody GAPDH ( I will not use the NAC well for Western blot because no need for it ) Please note that I can attach the results if you need it . In addition, I have to perform also Q- PCR to complete part 1 of the research. Part 2 , I should inhibit G6PD using DHEA or ShRNA. Also, 2 months ago I started to receive Prostate Cancer tissues from patients, I am growing each sample on 60mm X 15mm treated plate and this time I am waiting for the cells to grow from the tissues after cutting them into 1mm small pieces, as I am thinking of using the primary prostate cancer cells NOT fibroblasts this time to repeat all the methods again on PC primary cells. But because the cells are growing very very slow so I may not be able to wait until the cells grow up-to 80% confluence on the plate so I don’t think I can perform the experiments on them. Therefore, I purchased LnCaP cells and I will receive them next week . Then later I can re-do all the experiments again on them after I finish part 1 and 2 on fibroblasts. I have not start writing yet , so I firstly need now to write a good strong abstract for my thesis. Next, analyze the data and the results by using FlowJo or any other program ( no specific program is required) for the experiments that required Flow Cytometry. And explain and organize all the other done/ listed experiments. The most importantly is to use program that can open the results that are taken from the machine : Flow Cytometry . The software that is installed in the computer that is connected to the machine to show the results is called : CytoExpert. I copy the results from this software to a CD then I install it into my personal computer. So enable to open these dot blots and histograms you need to have a software that can open the results taken from the CytoExpert. And then, you can start combining the histograms for 1 experiment into 1 figure and then write an explanation: explain the results. Illustrate the purpose of all the experiment done and its illustrate its results. No limitation for the number of pages for the analysis. Also, no limits for the references but the number of words for abstract as any other thesis’s abstract usually around 250 words or a little more or 1 to 2 pages maximum. If you need further explanation please let me know. Thank you ! I attached the CCK8 results Please note that I measured the plate after I add the medicine after 14 hours, then after 24 hours , then after 36 hours, then after 58 hours , then after 72 hours. All the results are in this excel sheet. Thanks — https://www.transferbigfiles.com/e1e3638e-a446-4535-9df8-67da5ae1a069/YiMTR_1E7S3sAhqGCiPEoA2 share transfer: http://tbf.me/a/B0DZwl I uploaded here the ROS experiment results. these histograms are taken from the software CytoExpert so CytoExpert can open these histograms. Also, the software Flowjo will open it and analyze it. Please forward the email with the description to the writer. >>> To take these images I used the Microscope: Observer Z1( Axio observer Z1) and I convert the images to TIF so you can open it . I used 6 wells plate and 5 wells only of this plate has been used because I used only 4 different concentrations : 4mg/ml , 2 mg/ml , 1 mg/ml , 0mg/ml ( which I didn’t add the medicine to it ) and the NAC well ( which i add to it 20ul of N-acetyl- L-cysteine of 0.5mol/l – dissolved in Medium- to wash the ROS . Incubated it For 1 hour at 37 C then washed the well with incomplete medium then add to it 2 ml of medium mixed with the 2mg/ml medicine concentration as we found that this is the optimal concentration of medicine. Then incubate the plate for 48 hours and then after the 48 hours I followed the DAPI protocol and then took these images. >>>> Due to the large size of each image so I will be attaching each email with each concentration of medicine ( the >>>> concentration’s images) in a seperate email. >>>> In this email, the concentrations of : 0mg/ml mages are attached. >>> >>> Each well/each concentration has 3 different images. >>>> >>>> thanks — share transfer : http://tbf.me/a/EYrP1 https://www.transferbigfiles.com/7726081a-5a92-47bc-af44-155c3c23368c/LBb78mgtDiTxRzXBD6aStg2 here is the JC-1 experiment results. as you can see the negative control is wrong. Also as ROS, I used the machine Flow Cytometry to get the results, the software that is installed in the computer that is connected to the Flow machine is called : CytoExpert. So you can use the Flowjo software to analyze the data share transfer : http://tbf.me/a/CHv7Gt https://www.transferbigfiles.com/1a42542a-4259-4fab-8de3-3055e3d87f3d/XI-Va1EhpaV_DZtCW0ztJA2 here I uploaded the Apoptosis experiment results. the same like ROS and JC-1 experiment needs the FlowJo to open it and analyze the data. or any similar software. share transfer : http://tbf.me/a/VqNk2 https://www.transferbigfiles.com/4c58aaf2-18d6-454c-bd0c-bcea87200be9/yhCmml2vOhk5d1oJZ3duLA2 here i attached DAPI experiment results for the Nicotinamide concentrations : 0mg/ml which has no medicine added, and 1mg/ml concentration. I already sent the email with these images to the customer support with the description to send it to the writer but i attached here in TransferBigFiles again for confirmation. share transfer : http://tbf.me/a/BdMJFi https://www.transferbigfiles.com/e0db0e5b-e192-42bb-9562-18642da81dd7/6TFm37QkUy0k8vRS7LPlGA2 these are the rest of the images for DAPI experiment for the medicine concentrations of 4mg/ml and 2mg/ml

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