Has anyone successfully cultured oral epithelial cells (Buccal cells) from cheekswabs?

I'm a graduate student and I have collected cells from cheekswabs of donors. I have them in bronchial epithelial cell growth media in adherent T25 flasks. The cells look healthy even after three...

09 October 2017 6,134 1 View

Pre-amplification using TaqMan gene expression assay?

Can I pre-amplify a gene of interest using TaqMan gene expression assay for that particular gene from Thermo? If so, do I have to use their TaqMan pre-amp master mix, which is $900 or can I use...

04 May 2017 1,405 2 View

TF overexpression in IHC and western but gene expression data shows otherwise?

I'm studying a transcription factor in our patient tumor tissues. I have done IHC on about 25 patient tumor tissues vs. adjacent normal tissues. IHC and western shows a significantly more...

10 November 2016 7,800 4 View

Why do I have to make sure nuclei is intact when isolating plasma membrane protein?

So I'm learning that in order to increase the yield of membrane proteins by biotinylation and strep-magnetic bead pull-down, I must make sure nuclei is intact after lysis, therefore using...

05 June 2016 4,934 2 View

Difference between NHS-LC and NHS-SS biotin?

I'm trying to biotinylate surface membrane proteins with NHS-LC-Biotin with Strep-magnetic beads. I'm having trouble with identifying membrane protein with MS (very few identified protein and only...

05 June 2016 5,175 5 View

Phosphatase inhibitors or sodium orthovanadate?

I'm trying to isolate phosphopeptides from flash frozen samples. A sample prep protocol uses sodium orthovanadate as phosphatase inhibitor. Can I use commercially available phosphatase inhibitor...

03 April 2016 1,437 3 View

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02 March 2021 3,060 3 View

Should we change media after1st 6hrs after plating HUVEC bought from INVITROGEN?it's not recomended by invitrogen.Will DMSO present in stock matter?

We have HUVEC bought from Invitrogen. Cat no -C-003-5c. while first plating the cells we have bought the protocol suggests not to centrifuge the cell with media to remove the DMSO. Should we...

02 March 2021 3,713 2 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

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28 February 2021 9,936 2 View

Bought HUVEC from Invitrgen.need to revive it.expected it is cryofreezed in DMSO.Do I need to remove the DMSO for plating the cell for the 1st time?

After I cryofreeze the cell do I need to remove the DMSO for culturing the cell for next passages .

28 February 2021 7,738 3 View