2 replies to this topic

[Celltweety]

Hello,

I hve been performing PBMC culture for MIF protein analyses. I cultured PBMC with PHA and IL2 for 3 days and collected the cell supernatant and medium.
I performed a sandwich elisa for MIF protein. I have got high levels of MIF in 0hr lysate sample followed by a sharp decrease in 24hr for both control and treated cells. Then the PHA treated cells starts incresing in 48h and 72h when compared to control.

In medium samples the control cells are having high MIF concn than PHA treated.

What would be the reason behind this? Is it that the MIF gets synthesized inside the cell till 72h and then released or degraded as per the needs or how it goes?

Does anyone have a idea of how the secretion occurs. We also studied the gene expression wherein we could'nt find any drastic change in 3 days MIF samples.  

Thanks in advance
Celltweety

[almost a doctor]

Celltweety, on Aug 20 2009, 09:07 AM, said:

Hello,

I hve been performing PBMC culture for MIF protein analyses. I cultured PBMC with PHA and IL2 for 3 days and collected the cell supernatant and medium.
I performed a sandwich elisa for MIF protein. I have got high levels of MIF in 0hr lysate sample followed by a sharp decrease in 24hr for both control and treated cells. Then the PHA treated cells starts incresing in 48h and 72h when compared to control.

In medium samples the control cells are having high MIF concn than PHA treated.

What would be the reason behind this? Is it that the MIF gets synthesized inside the cell till 72h and then released or degraded as per the needs or how it goes?

Does anyone have a idea of how the secretion occurs. We also studied the gene expression wherein we could'nt find any drastic change in 3 days MIF samples.  

Thanks in advance
Celltweety

Hi celltweety,  a couple of questions.

1st: I'm not sure what you mean by "cell supernatant and medium", isn’t that the same thing?
2nd: What do you mean by "0hr lysate". Are you collecting the supernatant (secreted protein) and then lysing the cells (intracellular content)?  

MIF is constitutively expressed in most cell types, and as such you don’t need to stimulate the cells to detect this protein if you analyse the lysate, and will most probably not see a difference in protein levels.  MIF is only secreted after stimulation; the cells will secret different levels of MIF depending on the stimuli they receive.  For this reason you should only analyse the supernatant if you want to analyse the effect of your stimuli on MIF secretion. Same applies for RT-qPCR, is very hard to detect any differences in expression at the mRNA level of this protein.
MIF secretion levels are highly dependent on the inflammatory environment, and although it is supposed to be a type 1 cytokine, is also expressed and induced in type 2 settings. Finally, MIF induces secretion of itself, as well as of other pro-inflammatory cytokines (such as TNFa and IL12) which will in turn induce more MIF secretion.

Hope this helps and is not too confusing, MIF is a rather interesting, if very confusing, cytokine  

[Celltweety]

almost a doctor, on Aug 20 2009, 01:22 AM, said:

Celltweety, on Aug 20 2009, 09:07 AM, said:

Hello,

I hve been performing PBMC culture for MIF protein analyses. I cultured PBMC with PHA and IL2 for 3 days and collected the cell supernatant and medium.
I performed a sandwich elisa for MIF protein. I have got high levels of MIF in 0hr lysate sample followed by a sharp decrease in 24hr for both control and treated cells. Then the PHA treated cells starts incresing in 48h and 72h when compared to control.

In medium samples the control cells are having high MIF concn than PHA treated.

What would be the reason behind this? Is it that the MIF gets synthesized inside the cell till 72h and then released or degraded as per the needs or how it goes?

Does anyone have a idea of how the secretion occurs. We also studied the gene expression wherein we could'nt find any drastic change in 3 days MIF samples.  

Thanks in advance
Celltweety

Hi celltweety,  a couple of questions.

1st: I'm not sure what you mean by "cell supernatant and medium", isn’t that the same thing?
2nd: What do you mean by "0hr lysate". Are you collecting the supernatant (secreted protein) and then lysing the cells (intracellular content)?  

MIF is constitutively expressed in most cell types, and as such you don’t need to stimulate the cells to detect this protein if you analyse the lysate, and will most probably not see a difference in protein levels.  MIF is only secreted after stimulation; the cells will secret different levels of MIF depending on the stimuli they receive.  For this reason you should only analyse the supernatant if you want to analyse the effect of your stimuli on MIF secretion. Same applies for RT-qPCR, is very hard to detect any differences in expression at the mRNA level of this protein.
MIF secretion levels are highly dependent on the inflammatory environment, and although it is supposed to be a type 1 cytokine, is also expressed and induced in type 2 settings. Finally, MIF induces secretion of itself, as well as of other pro-inflammatory cytokines (such as TNFa and IL12) which will in turn induce more MIF secretion.

Hope this helps and is not too confusing, MIF is a rather interesting, if very confusing, cytokine  

Hi,

Thankyou for your inputs.

Yes the  cell supernatant and the medium are the same (i have written wrongly).
I collected both PBMC cells all the 3 days and its corresponding medium (supernatant). I use the medium and lysed cells for ELISA. We use both just to make sure if the MIF is in high levels in medium or inside the cells after stimulation. Also, the samples i am using here are just normal subjects. so there is less chance of vigorous inflammatory response present.
As you say i should have got a high value of MIF concn in medium samples, whereas my results show that MIF is more in cell lysates than in cell supernatants.
Does PHA+IL2 though stimulate the MIF production but at the same time it stays inside the cell for atleast 3 days?
Sorry if i confuse you once again but i think there exists some other reason/mechanism for secretion of MIF from cells (PBMC's).