I think it is useless to incubate heat killed bacteria.

Muthanna Hadi Hussain Thank you for the answer. I am trying to observe that is there any stimulation from cell debris such as bacterial components or its DNA.

Do you have any suggestion for me about both of the above protocol?

Hi Kittapart Chantakorn

Several suggestions:

1. BDMS are often semi-adherent depending on how macrophages were generated from murine (i assume) bone marrow.

In our lab once BDMS are seeded, we incubate them overnight to facilitate the adhesion and let them rest before stimulations. If you seed them together with your stimuli, there's too many things happening at one time, so this could affect your responses.

2. If you stimulate with the bacteria for X amount of time, keeping consistent MOI is crucial. Since they are dead this should not overgrown so you can try low medium and high moi (1, 5, 10 bacteria to macrophage cells). Sometimes under stimulation or overstimulation dampens the responses so this has to be optimized.

3. You can for sure use a conditioned media for stimulation. Several suggestions:

a. if possible, use cell culture media for macrophages to grow your bacteria of interest.

If that's not available, you have to use a small amount of microbial media for stimulation, since the microbial media components often results in stimulation. Make sure to include sterile media control.

Same as MOI, you can use variable amount of conditined media for stimulation to find the best amount that induces your stimuli of interest.

Include the positive control to make sure your macrophages are reacting. Depending on your goal, this could be LPS+-Interferon gamma, PMA, PolyDT ant so on.

Best of luck,

Povilas