Can we derive organoids from 2D cell culture?

I am keen to derive organoids from my existing primary tumour cell culture. Is there any existing protocol that you would recommend?

02 March 2018 2,564 3 View

Why does shRNA-mediated silencing have different effects on cell signalling as compared to that by siRNA-mediated silencing?

I started off by creating a stable knockdown model using a control shRNA and two shRNA specific for my gene-of-interest. I achieved 90% knockdown after antibiotic selection, and the knockdown...

07 August 2014 7,765 7 View

Does anyone have any suggestions on a comet assay for DNA damage and chemoresistance testing?

I would like to use a comet assay to test the effect of my protein-of-interest on DNA damage response, double strand breaks and chemoresistance in cancer cells. There are several types of comet...

05 June 2014 7,838 3 View

Can anyone help with loading controls for high molecular weight proteins?

My proteins of interests are ~350-500kDa. The usual loading controls have much lower molecular weights. I run 6% (non-gradient) gels. Are there any suitable loading controls out there?

01 February 2014 1,434 5 View

Is vinculin an ideal loading control for large proteins?

I recently started using vinculin as a loading control for high molecular weight proteins. Strangely, vinculin showed up evenly despite a 2-fold difference in loading. I wonder what is happening...

01 February 2014 8,671 3 View

If protein of interest is at undetectable mRNA level, is knockdown data still valid?

A protein of interest has an undetectable mRNA expression level in my cell line. I knockdown using siRNA (and overexpressed, but that's a different story) and surprisingly was able to detect the...

06 July 2013 972 8 View

No title

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

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

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

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

No title

28 February 2021 9,936 2 View

How to compare 2 samples recruited with different sampling methods?

If I want to compare characteristics of 2 groups of patients, one group is recruited from a random sample of the general public while another group is from a sample with a fixed gender (male to...

26 February 2021 3,526 3 View

Has anyone had luck using cell lines other than HEK 293 (such as COS1) to package lentivirus?

I am looking to package lentivirus in endothelial cells but I am opposed to using 293 cells due to personal ethics regarding the source. I have done some reading and saw that COS1, COS7, & CHO...

25 February 2021 3,030 1 View