CMIRC

Dear All

I need some help. I am working with complement protein iC3b and need to heat inactivate Normal Human Serum (NHS) to inactivate iC3b as a control. However, it is not working. At the moment I heat NHS at 56 degrees for 60 min and then freeze it for storage in -80 freezer. Whenever I use it for complement assays and label cells with the complement antibody I get binding. This therefore means that the NHS has not been inactivated totally. I have tried the conventional method of heating at 56 degrees for 30 min but even that did not work. I would therefore like to find out a quick way to test if the NHS has been heat inactivated.

I am open to suggestions.

I am trying to induce early apoptosis in PC3m cells and K562 cells using heat induction. However I have been unsuccessful. I either get viable cells or dead cells and nothing in between. My method in brief is suspending 250 000 - 500 000 cells per eppendorf microfuge tube in 100 ul of RPMI. I then incubate the cells in a water bath preheated at 43 degrees celcius for 40, 30, 15, 10 mins. After incubation I check for it using Viability reagent using the Flow cytometer. However with both the cell lines I either get high viability or high cell death.

I would be greatly appreciated if somebody can offer me some advice on how to achieve early apoptosis or even late apoptosis. A protocol that has been tried and tested would be good, however I am open to all suggestions.

Hope to hear from someone soon

CMIRC

Dear All

I am confused as to how cell passaging works. My question is:

If I thaw a vial of PC3 cells (prostate cancer immortal cell line) and culture them. The passage number would be 0 when I thaw them first. However the passage number would increase to 1 when I sub culture them. Say after subsequent cell culturing I decide to freeze the cells at passage 4. The next time I take out a the PC3 vial from liquid nitrogen and culture would the passage start from 4 or from 0?

I know that for primary cell lines the passage number does not reset and continous even after freezing and thawing. however does the same apply for immortal cancer cells. And if it does then upto what passage can one do experiments without having any genetic alterations due to the passaging. ie upto what passage can one use an immortal cell line before a buying a new one?

Also for non adherent cell lines when would a passage be considered. If I take flask of 50% confluent THP1 cells and centrifuge them and put them back in the same flask with new media without splitting would that be a passage, secondly if I have 2 flasks of THP1 cells 25% confluence and I put them into one flask without changing the medium or centrifuging, then would that be a passage?

Hope to hear from somebody soon.

CMIRC

I am new in qPCR and have recently got this kit that requires instrument specific optical qPCR plates. The instrument we have in the lab is the MJ research Opticon 2 machine. As far as I am aware it only takes 96 well plates. Checked online on various websites: fisher, sigma. However they all have qPCR plates that range from different colours to different types like skirted or unskirted. Can anyone advice me as to which kind of plate in terms of colour and skirting I need for the qPCR?

Hope to hear from anyone soon.