Hi Sreeram,

For flow cytometric analyses, we pretreated RAW264.7 cells (1 × 106cells/ml) with medium, CpG DNA (0.06–3 μg/ml), or non-CpG DNA (0.06–3 μg/ml) for 24 h. After pretreatment, cells (5 × 105 cells/ml for TNF-α, and 1 × 106 cells/ml for IL-6, IL-12, and IL-12) were stimulated with medium or LPS (50 ng/ml) for 6 h (for TNF-α) or 24 h (for IL-6 and IL-12, B and). The levels of TNF-α, IL-6, or IL-12 in culture supernatants were determined by ELISA.

Generally, for intracellular staining, we also used the above cells incubated in Cytofix/Cytoperm reagent for 15 min at room temperature and then stained with PE-conjugated anti-mouse TLR4/MD2 or anti-TLR9, followed by staining with FITC-conjugated anti-mouse IgG1. Stained cells were analyzed on an EPICS XL-MCL using software like EXPO32 ADC. 

Best wishes,

Ilya

Hi Sreeram,

During stimulation we add 10ug/ ml Brefeldin A to stop cytokine secretion. Following stimulation, first stain with antibodies for surface markers (20min 4C), wash, fix with 1% paraformaldehyde (20 mins dark RT), wash and then stain with antibodies for any intracellular proteins (including any cytokines) in the presence of 0.4% saponin (4C 30min or overnight). Wash and then run on a FACs machine. From what I've seen you should be able to analyse all three cytokines in the same reaction based on different antibody conjugates.

Good luck,

Damien

Sreeram,

    There are two things you need to think of first:

- use Fc blocker to avoid non-specific staining. Macrophages express high levels of Fc receptors.

- exclude dead cells with a Live-DEAD marker

  Now you can look at your intracellular cytokines (adding Brefeldin A with your stimulus will help increase the amount of detectable cytokines within your RAW264.7 cells; but Brefeldin A is toxic to the cells long term). No reason this shouldn't work.

Thank you Ilya, Damien and Eric.

Damien: May I ask if the protocol you have recommended was tested specifically on RAW264.7 cells? Thank you...