Need for Help (DC, IL12,LPS, autoMACS)

[Masoud]

Dear friends

In my research i need to isolated DCs in popliteal lymph-nodes and then culture them by LPS and then IL12 assay by Elisa.

I used Anti CD11c magnetic beads and autoMACS to isolate DCs.

My Elisa results shows even in culture of DCs with No Ag we can detect high level of IL12 production!

Is it possible it be for magnetic beads??

any recommendation? Using cell sorter is not practical in this case because cell number is very low.

hope to hear you soon.

Thank you very much

Masoud Akbari

Nagasaki University

Japan

[dust mate]

Dear friends

In my research i need to isolated DCs in popliteal lymph-nodes and then culture them by LPS and then IL12 assay by Elisa.

I used Anti CD11c magnetic beads and autoMACS to isolate DCs.

My Elisa results shows even in culture of DCs with No Ag we can detect high level of IL12 production!

Is it possible it be for magnetic beads??

any recommendation? Using cell sorter is not practical in this case because cell number is very low.

hope to hear you soon.

Thank you very much

Masoud Akbari

Nagasaki University

Japan

 

Can not you perform intracellular staining after culture of whole popliteal LN cells instead of purification of DCs? I am not sure even with CD11c MACS you will achieve high purity anyway. these beads work well when initial cell frequency is high (e.g. in the spleen after 2 rounds of AutoMACS I was getting around 60-70% CD11c+ cells (which is low) when initial frequency was 1-3% only. but when initial frequency was around 30% then purity was >97%).