How long LPS stimulation of monocytes?

[Paja]

Hi everybody,
I am wondering how much time does it take to stimulate peripheral blood monocytes by LPS to see switch in CD markers (from CD14++ to CD14+/CD16+ or CD16++) and other changes in expression of CD80, CD86, CD69 etc. I have already learned I have to stimulate cells by LPS at least 12 better 24 hours to detect cytokine production in supernatants, but how quickly do CD markers react?

Thank you for your kind advices.

Paja

[Deepak Nayak]

Hello Paja,
Monocyte/Macrophage maturation by LPS is usually pretty quick and by the 12 or 24 hr when you assay for cytokines in the supernatant all of these markers should be on. I would titrate LPS in nanogram/mL range and in 24 hrs post treatment surface stain for the co-stimulatory molecules. In the most probable case, the cells will be really sticky and scraping may be necessary to get them off the flask. Additionally, I would recommend to include a anti-MHC II probe to make sure that both class II and co-stims are turned on. Since you are also going to analyze for FcR, it is essential to include a blocking step with Fc-block to reduce background.
Let me know if this helps.
DN

[Paja]

... Since you are also going to analyze for FcR, it is essential to include a blocking step with Fc-block to reduce background.
Let me know if this helps.
DN

Hallo Deepak Nayak,
Thank you for your response. Unfortunatelly I had to cut this issue off for a while (several months) in order to complete other projects and that's why I react to your post with such a delay. What I meant in my original question was not only what is the best time period for stimulation to be able to detect changes in ALL mentioned markers (CD80, CD86, CD69 and yes, we plan to include also HLA-DR), but also, if some of them does peak much earlier than the others (i.e. after 4 hours of stimulation) with subsequent decrease, so that after i.e. 24 hours, when I want to check for all markers, this one will be back at zero (or pretty low)... Such a situation, of course, makes me to do false interpretation. So, if this is the case, I would have to stop stimulation after 4 hours to assay for early activation marker and than after 24 hours to analyze late activation markers...
And second question of mine concerns Fc receptor blocking: I do not understand your note about it: When I am going to detect Fc receptor on monocytes, I thought I have to OMIT using BSA or FBS for blocking these receptors since than my anti-FcR Ab can not bind... Am I missing somtehing? This blocking and non-specific binding issue is sort of weird for me...
Thank you once more,
Paja