I am totally new to research, i am an undergrad. I need some help!

I am trying to do intracellular staining of isolated T cells. I do not want to induce cytokine production with PMA/ionomycin however. I DO want to use monensin to prevent exocytosis of the cytokines already present!

How should I go about doing this? All the protocols I have found just say to add the monesin with the PMA, but i dont want to use the PMA.

ALSO, should i even be using monensin for IL-4 and IFN-gamma, or does brefeldin A work better?

I have not had success so far, while not using a protein transport blocker. I am hoping that the transport blocker is going to make a huge difference.

can anyone help? THANKS

I think your just going to have to try it without the PMA+IO using your protocol and see if you have enough signal to measure. Depending on which cytokines your looking at, this may or may not work. It can be difficult to measure sufficient levels of cytokine production even with stimulation. Just skip the stimulation and go to the next step with the transport blocker. Generally the blocker is added for around 4-6 hours, but this something you may want to titrate for yourself to optimize. I haven't compared the two blockers, but I'm not surprised that you didn't see anything without using any blocking at all.

You may find this reference useful: Pala, P., T. Hussell, and P. J. Openshaw. 2000. Flow cytometric measurement of intracellular cytokines. J Immunol Methods 243:107-124.

Hi! Since your PM had essentially the same questions, I'll try to answer them here, where its indexed.

>>
I am trying to do intracellular staining of isolated T cells. I do not want to induce cytokine production with PMA/ionomycin however. I DO want to use monensin to prevent exocytosis of the cytokines already present!

>>Baseline cytokine levels in resting cells are VERY low - I've had some patchy results even detecting anything above isotype. Your best bet is to make cell lysates and run Westerns or perform a sandwich ELISA (remember to make a non-denaturing lysate for ELISA, standard Laemmli SDS lysate works for Westerns, 15% gel, PVDF transfer.)

How should I go about doing this? All the protocols I have found just say to add the monesin with the PMA, but i dont want to use the PMA.
>>The PMA (or PMA+Ionomycin) serves to activate cells, and enhance levels of cytokines. If your cells are already pre-activated (as opposed to naive, lets say owing to ova priming+challenge), they will produce more cytokine.

ALSO, should i even be using monensin for IL-4 and IFN-gamma, or does brefeldin A work better?

>>This cytokine produced (or already present in the cell) is still at fairly low concentrations. Monensin or BFA help tilt the odds in yourfavor, to enable detection. Both work in a similar manner (ER-to-Golgi transport or Cis-median Golgi transport is inhibited, preventing export of protein), both are toxic to cells if left on too long (typically BFA is 2-6 h and monensin is 4-8 h). Either worksfine - monensin is cheaper and more stable, BFA is slightly more efficient (hence the shorter incubation time - its also more toxic).

I have not had success so far, while not using a protein transport blocker. I am hoping that the transport blocker is going to make a huge difference.

>>All the best! If you are using BFA, get the 1000x stock from eBioscience. If using monensin, use the powder from Sigma.I do believe i put up a protocol for intracellular cytokine staining from my lab - its loosely based on the BD Pharmingen FACS protocol, with modifications to use our own buffers.

THANKS guys!!

before i posted this I ordered monensin from BioLegend
http://www.biolegend.com/index.php?page=pro_detail&cat=-1&detail=1500

hopefully it will help!


....


There is another problem I would like to ask you guys about. After I permeabilize my cells with Triton x-100 .2% for about 10 minutes, I have WAY less cells when I run the samples in flow. I then tried .1% and still had WAY less cells, as I ran a control that was not permeablized.

Is it better to fix the cells prior to permeablizing? (i just started doing that)

Also, how important is the g force when spinning? I was spinning at 3000 rpm for 3 minutes. I am not sure what g force that is, but i do know that 1800 rpm is 300 g's on our centrifuge. Could I be spinning to fast?

ONE more thing, how important is it to purify the T cells? I usually have lots of red blood cells in my isolations. Should I be doing a density gradient to extract the lymphocytes? (i did one once and i think it might have helped)



THANKS for any help!!!!

FIX the cells FIRST - before you permeabilize (after any surface staining is complete)! Fix with 4% paraformaldehyde, pH 7.2-7.4 or 10% neutral buffered formalin (NBF - Sigma)

Permeabilize with 0.1%Triton X100+0.5% lowIg serum+HBSS/PBS.
Triton x100 can be replaced with FRESH 0.3% saponin (starting from the white powder, not the brown one). Saponin gives better results (less bkgrnd), but is cumbersome toprepare/store (gets oxidized to the brown form very easily).

For lymphocyte suspensions, 330 g / 5 min (~1250 rpm in most std swing out rotors in Beckman benchtop centrifuges) is ample.

I always do a density gradient - that also gets rid of most RBCs without using a RBC lysis solution (You can make your own or get it from BioLegend - its ~$40 for 100 ml of 5x).
Use Ficoll 1083 from Sigma or similar, as gradient medium for for mouse LN/spleen.

If you also concurrently surface stain for CD3, CD4 CD8 in a multi color FACS, no additional purification is required unless you want to do certain kinds of functional assays ex vivo.