No, I do not have.

Dear Miriam 

For how long are you treating the macrophages?.

Best,

Jaime

Can you see vacuolation by phase contrast?. Is there any change compared to control treated macrophages?

Maybe it's a matter of the dose of oxLDL you are using. Have you checked the levels of CD36 on the surface of your macrophages?, maybe there is no internalization. Perhaps you can try in serum free conditions for a short time.

Good luck!!!!!

Hi Miriam!

I haven´t worked so much with oxLDL & foam cell formation, but there's a long history at our research institute. I hear 200 ug oxLDL /ml medium for 48 h should work fine. We have used mostly copper-oxidized LDL.

Could the positive oild-red-o staining in your untreated control cells be related to culture conditions before the oxLDL loading exp (or during)? Do you culture your cells / perform your experiments in the presence of serum?

We culture our primary human monocytes for 7 d in Gibco Serum-Fee Macrophage Medium supplemented with antibiotic and M-CSF to induce macrophage differentiation. The serum-free culture system was initiated, because people working with lipoproteins wanted to avoid the introduction of an undefined set of lipoproteins derived from the serum preparations, a factor that might interfere with the subsequent LDL loading & efflux experiments.

I hope this was helpful, I wish you all the best with the experiments!

Miriam,

oxLDL preparations are unstable on time. You should better try acetylated LDL. Acetylation is not difficult to achieve just incubate your LDL with acetic anhydride in a saturated acetate solution. 

When stimulating foam cell formation try to avoid serum in the medium. we use a medium supplemented with albumin and glucose (besides the ATB).

Hope you get it through!

Hello Miriam !

Several months ago I had the same question to this audiance. I asked for a working protocol but nobody was able to share his experience with me, instead, I receive a couple of unhelpfull questions . I stopped working on this because it was very frustrating, time and money consuming. You received a lot more suggestions than me so maybe you can generate successfull foam cells. I would be very glad if you could share your experience (maybe you generate a one-to-one protocol for "dummies" ?) or even cooperate with me. I would be very pleased if you could stay in contact with me ([email protected]).

Good luck, Atilla

Miriam,

Here is how we prepare Acetilated LDL. All the protocol is made on ice. Dilute LDL to a concentration of  40 mg of proteins / dl in PBS.  Then add drop by drop 1ml of a saturated Sodium acetate solution / 10 mg of total proteins (total mass of proteins). After mixing gently add 2,5 uL of acetic anhydride / mg of total protein mass. The total volume of anhydride you have to add must be divided in 5 aliquots which will be added every 15 minutes. (Total incubation time 1h15min). To stop the acetilation dialize the modified LDLs ON.

Before doing the reaction save an aliquot of the original LDL preparation to check for the extent of acetilation. Use a lipoprotein electrophoresis assay to check how the reaction worked out. Ac-LDL run faster than native LDL.

Hope this will help you out.

 Dear Miriam

One question, what is the source of your LDL?, if it's human and out of curiosity, can you get LDL from patients suffering from heart disease/atherosclerosis and try them for phagocytosis with your macrophages?. LDL particles from these patients should be highly oxidized and maybe they are highly inflammatory/phagocytic.

Best,

Jaime Llodra