hello,

using of serum has a great impact on phagocytosis, the serum complement component and the serum fibrinogen also have very important role in the phagocytic process of the macrophages through complement recetors (CR1, CR3,CR4) besides, a significant differences can also be found  between the using fresh and frozen serum, i was working on the role of different host sera (human, chicken and FCS) in M. avium invasion of human and chicken cells. as well i compared between fresh and frozen serum.

Changing and washing of the seeded cells are also very important, firstly to refresh your cell before invasion and to keep you cell growing well as acidic ph of the media will affect on your cell viability especially if you are planing to incubate your cells more than 24h.     

Thanks a lot for the reply.

So is it good to work with non heat inactivated serum or it's better to use HI serum? Especially using Fluospheres?

Thanks a lot in advance,

Alice

to detect the role of the serum components in the invasion rate is preferable to use the non heat inactivated serum, but if you want to use the serum just to provide the cells with the required nutrients it dose not matter whether to use the HI or non activated one 

regards

Thanks a lot.

By the way, the effect of heat inactivated serum depends on the species and on the size of the phagocyted particles as well.

About species: the heat inactivation counts less in mice PMN and in human and mice monocytes/m1 macrophages, but will have a larger impact on human PMN (up 50.80 %). In my experiments, the heat inactivated opsonization resulted aprox. in 10-20% slower phagogytosis, but - after reaching the platetau - there was no diffferencies within the amount of phagocyted particles or in the number of phagocyting cells.

The size and concentration of the phagocyting particles also matters, I have experience in the range from bacteria size (800nm) to zymosan (up to 2 um), and the phagocytosis of the smaller sizes was faster, normalized either to surface or to concentration of the particles. 

On the other hand, there was no difference in fluorescent activity within the opsonizations when I use fluoresent labelling (e.g. antiFc Fab, or streptavidin-biotin), but after the phagocytosis the fluoresent signal was decreasing in a few minutes (probably due to the pH changes in the phagosome?). On the other hand, internally labelled particles (e.g. bacteria with Cell Tracker, or USA 300 S.aureus) were much stable in following the phagocyted particles on the base of fluorescent intensity.

I hope, it can help You! :) With bests!

Thanks a lot.

Alice

Weelocome, if it was helpfull.

Nawzat Aboziad Issa are we talking about fibrinogen added to serum or you meant plasma? Because serum should be free from fibrinogen and the phagocytosis observed in serum-condition shouldn't be accounted to fibrinogen activity. I find this a very interesting topic. Do you know what is the contribution of fibrinogen alone in phagocytosis?