43 Questions 26 Answers 0 Followers
Questions related from Jeff Zaki
hi i am not sure if it is alright to analyse my data this way. So i prepared 3 samples of controls, 3 samples of condition at 1hr exposure, 3 samples of condition at 6hr of exposure. i collected...
15 August 2017 8,803 4 View
i am aware that gene expression profiles might differ between cell types and types of mechanical forces. But what are some 'consensus genes' which expression level changes (high chance) upon...
20 June 2017 3,633 1 View
hi im a new researcher. i did qPCR recently, looking at gene expression change in certain genes. basically in a plate, i ran 3 biological repeat controls, and 3 biological repeat mechanically...
29 May 2017 5,825 4 View
hi i ran qPCR today, looking at gene 1 and housekeeping gene in a single qPCR plate. In this one plate, i ran all 3 biological replicates, ie: 3 biological replicates of condition, 3 biological...
27 May 2017 237 2 View
hi there. I am in a major hurdle block and I hope to get advise on this. My experiment involves putting mechanical stress on cells at different durations and looking at whether a particular...
24 May 2017 5,185 7 View
We usually use this kit which has a qia shredder column to lyse the cell before transferring the lysate onto another column for further RNA purification. However we have run out of this qia...
23 May 2017 976 3 View
hi i am currently deciding on whether to use DNA microarray or RNA-seq for my project. I am interested in studying the mechanosensitive genes: genes which expression level changes upon mechanical...
22 May 2017 3,858 9 View
hi i am applying mechanical force on my cells and would like to know if you have any tools available for looking at actin stress fiber length, thickness etc. imageJ plugins eg.
08 May 2017 2,889 0 View
hi i am currently looking at cytoskeleton changes of cell upon applying compression on cells. I have captured images of the stress fibers. I am pretty new to research. What aspect of the stress...
08 May 2017 1,343 1 View
hi this is the first time im detecting phosphoproteins. This is the recipe for my lysis buffer: 250ul of 1M hepes 600ul of 5M NaCl 10ul of 1M MgCl2 20ul of 0.5M EGTA 200ul of 1M B-glycerol...
03 March 2017 9,631 3 View
hi I observed such a-tubulin distribution changes in control and flattened(compressed) MDA-MB-231 cells. do let me know if you disagree or find other differences. 1) Upon compression, the...
28 February 2017 865 7 View
i am applying compression into the transwell insert and this compression cup occupies all the surface area of insert thus i can only add minimal volume into the transwell insert, most media is...
23 February 2017 966 2 View
hi i have been doing immunofluorescence on transwell membrane inserts and have been getting suboptimal images. i fix and stain the cells in transwell insert, and when i mount them i cut the...
14 February 2017 1,696 5 View
recipe for 10% PFA: add 25g PFA to 250ml water . Heat at 50 degrees till dissolve. diluting 10% PFA to 4% PFA: 10ml of 10% PFA + 2.5ml 10X PBS + 12.5ul of 20% triton + 12.375ml H2O. sometimes i...
09 February 2017 5,434 1 View
i did nuclear lamin staining on transwell insert which contain monolayer of MDA-MB-231 cells that were mechanically compressed. do you see any nuclear abnormalities? red: actin, green: nuclear...
03 February 2017 1,259 2 View
i did mechanical compression on cancer cells and then looked at a-tubulin staining pattern. I lack experience and did not see any changes in a-tubulin pattern. Do you see any changes? top image...
29 January 2017 437 4 View
i did mechanical compression on cancer cells and then looked at actin staining pattern. I lack experience and did not see any changes in actin pattern. it could take a keen eye to detect changes...
29 January 2017 893 3 View
hi i am thinking of doing RNA-seq on cells that are mechanically compressed. but before i do that, i have been advised to maybe look at RT-PCR for some genes first. what are some good genes whose...
23 January 2017 7,471 3 View
Hi I usually perform immunofluorescence on cells grown on coverslips. But this time im growing them on transwell inserts. All the fixing and antibody staining/wash steps will be soaking the...
12 January 2017 6,986 5 View
Hi i would like to know if the supplier of transwell recommends a particular catalog number for collagen to coat transwell insert, is it OK if i use collagen from other companies for coating?...
11 January 2017 4,028 1 View
hi i will be performing global transcriptome analysis of monolayer of cells exposed to mechanical compression. but before i do this, i will do staining on cells to look at effect of compression...
09 January 2017 7,504 1 View
Hi i am pretty new to research but would like your opinion if its still wortwhile to do an RNA-seq on breast cancer cells exposed to compression forces. RNA-seq is more global and can detect more...
29 December 2016 1,526 3 View
hi im a new researcher and is hoping for some opinion on this. i am working on podocytes cells, which are part of the glomerulus of kidney. They are therefore exposed to glomerular pressure, and...
24 November 2016 9,528 2 View
hi, is there a rough guide or from your experience, an estimation of cell density being translated to cell confluency? i need this rough translation for initial seeding density.
23 November 2016 4,445 5 View
Hi i would like to know the general way of indicating passage number to cells. I have heard that some people label the cell passage number as zero upon receipt from company. while some would just...
22 November 2016 7,539 4 View
hi i need a second opinion as to whether my podocyte cell lines are differentiating. these are SVI podocytes and they proliferate at 33 degrees celcius, differentiate at 38 degrees celcius. It...
07 November 2016 7,907 3 View
has anyone had to deal with cell lines that proliferate at 33 degrees and differentiate at 37-38 degrees (14 days needed) celcius? The cell line im dealing with are SVI podocytes (CLS). i...
03 November 2016 3,262 7 View
hi im a beginner in lab work. i have been doing multiple runs of western blot to detect protein (be it protein of interest or housekeeping proteins) but i couldnt get a good band. i realise...
27 October 2016 4,958 10 View
Hi im trying to detect by western blot a nuclear protein. I have been trying yo do this with no success until i realised that nuclear proteins may require different way to prepare and detect....
21 October 2016 4,063 5 View
Hi, I´m a new researcher and I am trying to verify a particular cell line before I start experiments. I am looking at expression of certain key proteins specific to this cell line. I have been...
17 October 2016 881 5 View
hi im new to research so i will be happy to receive any advise on this matter. I have a new cell line with me and i am proceeding to ensure that this cell line is indeed what it is by detecting...
16 October 2016 4,084 3 View
hi im new to research and have been told to always prepare the 5% milk FRESH each time and not to reuse it for other days, ie: store leftovers at -20 deg and used on other days. but i realise...
16 October 2016 5,787 4 View
hi i would like to know if sterility or accidental addition of microbes during immunofluorescence part must be observed. The steps starting from taking out the culture plates from incubator,...
10 October 2016 8,762 3 View
hi im working with podocytes. I am intending to perform IF on these cells. But one puzzling thing happened. 6hrs post splitting the cells into the plate meant for immunoflorescence, i notice the...
05 October 2016 8,974 7 View
hi i intend to perform actin staining as well as focal adhesion staining of my cells. I am pretty new to research work. Focal adhesions are composed of several proteins in 1 complex. Which...
01 October 2016 5,446 1 View
hi I would like to know your expert opinion on this matter. I have been reading up on podocyte response to mechanical stretch. Most studies wanting to investigate genes upregulated/downregulated...
29 September 2016 1,509 1 View
hi I intend to expose my cells to mechanical stretching while in SILAC media. Im a pretty inexperienced researcher being an undergrad but what kind of downstream mass Spec analysis can i perform...
20 September 2016 4,561 2 View
Plan for podocytes cell line: · 1 vial contains 1.5ml, to a total of 2.25 x 106 cells · Passage number upon arrival is 23. Each batch can be used until passage 40 Thawing from...
09 September 2016 9,199 1 View
hi i intend to study the phosphoproteome changes upon exposure of a glomerulus cell to increase glucose concentration. diabetic conditions have been shown to induce apoptosis in these cells in...
05 September 2016 2,727 2 View
im currently working on podocyte cell lines. protocol recommends use of accutase to detach adherent cells. can this be replaced by trypsin?
24 August 2016 4,152 3 View
Is there a general rule to follow as far as number of nucleotides and the sequence is concerned? Or is there a strict rule to follow depending on restriction enzyme to be used? Buffer sequences...
18 July 2016 5,862 3 View
i read this part here from a research paper, ''If the mean percentages of inclusion between two experiments are distinct enough so that the standard deviations do not overlap, these values can be...
03 May 2016 1,280 4 View
I have transformed competent E.coli cells with a plasmid. I then plate these on agar plate with antibiotic ampicillin. I did a mistake once where I overincubated the plates for 24 hours or so and...
07 March 2016 7,687 3 View
