How to estimate pregnenolone in cell culture media spent using HPLC?

07 July 2013 1 4K Report

I have to estimate a steroid called pregenenolone in cell culture media spent.

We measured pregnenolone levels in culture media of PC-12 and SHSY 5Y cells successfully. Our protocol is as follows

The cells replace with 2 x 10 6 density in 6 well-plates. Methanol/acetic acid (v/v: 1x10 4/1) ratio is used as a mobile phase for determination of pregnenolone levels. Pregnenolone standard is prepared in methanol. The conditions of HPLC methods (for Schimadzu LC) ; (4.6x250mm C18 ; methanol/Acetic acid (v/v: 1x104/1); 2 mL/min; 30 ºC; UV-VIS detector, 200 nm, injection volume 100 µL)

Badges
Science topic

More Dhiraj Singh's questions See All

How to isolate leydig cells from rat?

In leydig cells isolation protocols, there is one step where density gradient of percoll is made. Can anyone help me by providing the detail protocol for making this density gradient? Or if...

07 August 2013 1,897 2 View

No title

02 March 2021 3,060 3 View

Is There Any Feasible Method To Test Fluorescent Compounds Other Than Fluorescence Spectrometers ??

Is There Any Feasible Method To Test The Efficiency Of Fluorescent Compounds Other Than UV Spectrometers ? Suggestions Would Be Appreciated !

02 March 2021 5,785 3 View

Why almost every element would be metallized in sufficiently high pressure and low temperature?

(This statement is from wikipedia, BTW). What is so special about metallic bond that is limiting high-P low-T state of a matter (as long as individual atoms exist, not white dwarf of neutron star...

02 March 2021 3,309 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

Any research papers for extraction of nutrient deprivation on macroalgae? Analysed by HPLC-UV and GC/MS. Possibly from macroalgae genus Rhizoclonium?

I am doing my thesis on the nutrient deprivation on the macroalgae Rhizoclonium sp. I will be analysing the levels over a period of roughly 5 days by HPLC-UV and GC/MS. I will be using seawater as...

28 February 2021 6,477 1 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