MTT plating density?

Hi, I was wondering, would it be reliable to compare two cell lines using MTT if I plate them at different densities? For instance, cell line 1 at 1000 cells per well (because it grows faster),...

06 July 2019 523 3 View

RNA bands are more intense than protein bands?

Hi guys, So I am working with two colorectal cell lines (HCT116 and HT29). I extracted RNA, converted to cDNA and ran PCR for my gene of interest. Then also extracted protein and did a WB for the...

05 June 2018 5,363 5 View

Can someone advice on the selection step of CRISPR?

Can anybody please explain how to do the selection step? I am currently growing my cells in 6 well plate and after puromycin treatment, I can see physical colonies (like the ones you see when you...

04 May 2018 6,961 1 View

Is IC50 consistent for different plating densities?

Hi guys,. I have done MTT assays with different drugs with 1000 or 2000 cells plated in 96 well plates. When I obtained my results, the line on the graph of cell viability against drug...

09 October 2017 8,879 5 View

PCR for NEIL3 gene?

HI guys, I have been trying to amplify NEIL3 gene in HCT116 and HT29 cells but no luck. I amplified GAPDH successfully but not NEIL3. Please has anyone had luck with NEIL3, if they can suggest...

09 October 2017 9,368 1 View

Why do you need sterile PBS for cell culture?

I have done cell cultures in the past (during undergraduate and master's) where I used 10x PBS (for rinsing cells) directly from the manufacturer's bottle without any issues. Now that I am doing a...

01 January 1970 6,957 3 View

Cell Growth Curve

Hi guys, I wanted to know, if I am conducting research experiments (such as transfection, MTT, etc) using 96 wells plates, would it still be ideal to generate my growth curve using 24 well plates?...

01 January 1970 2,787 2 View

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02 March 2021 3,060 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View