I encountered the problem of no signal detected from the flow analysis of my THP-1 derived dendritic cells (DCs). I have observed the morphology of my cells resembling that of DCs after differentiation (see attached photo). However when analyzed using flow, no signals could be generated. Here is how I prepared my cells:
1) Differentiate THP-1 (2x10^5 in 5mL) according to protocol (RPMI 1640 w/ phenol red + cytokines)
2) Wash the DCs with PBS w/o Ca2+ and Mg2+ and detach using 0.25% trypsin-EDTA.
3) Block with Fc Receptor Binding Inhibitor (20µL per 100µL) on ice for 20 mins.
4) Wash and incubate with a fluorophore-conjugated primary antibody against CD80 (from thermofisher 11-0809-42), at a dilution of 1:10 in commercial staining buffer, for either 1 hr at room temperature or 4°C overnight.
I am wondering which step have I gone wrong. Is the antibody used not enough? I initially suspected the incubation was not sufficient so I tried incubating overnight. Yet I got the same result with no signal. Does anyone have experience working with this cell line for flow analysis? Will appreciate it so much if anyone can help.
You could try scraping the cells rather than using trypsin just in case the trypsin is cleaving CD80. You could also grow them on a chamber slide and use the antibody for ICC to check this.
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