need a comprehensive protocol for isolating and culturing human monocytes - (Jan/25/2013 )

Could some who has done this procedure before share with me there protocol in detail please.

Hi Zac70,

here's the protocol I've used.

Collect samples into EDTA bottles. (I usually collected about 30ml of blood per person)

Place fresh blood samples into 15 or 50ml conical centrifuge tubes.

Slowly layer the blood over Histopaque 1077. (Use 3ml Histopaque per 7ml blood)

Centrifuge for 30min at 2000rpm at room temperature. Make sure no brake is applied.

Using a sterile pipette, remove the plasma layer and save for use at a later stage. Using another pipette, transfer the mononuclear cell layer to another centrifuge tube.

Wash cells by adding excess HBSS (3 times the volume of the mononuclear layer) and centrifuge for 10mins at 1300rpm.

Remove the supernatent, resuspend cells in HBSS, and wash twice more in a similar manner.

Suspend mononuclear cells in RPMI + 2% plasma/serum + Pen/Strep (i.e. what was removed prior to isolation of buffy coat).

Count cells and determine viability by trypan blue exclusion.

Seed cells appropriately and allow to adhere for 2 hours.

Check to ensure adherence.

Remove non-adherent cells by gently washing three times with RPMI + 2% serum + Pen/Strep and replace with 2mls of same media.

To generate fully differentiated monocyte-derived macrophages culture the adherent monocytes for 7 days. Change half the media after 4 days. Check purity by immunohistochemistry using CD68/CD14.

Hope this helps!