FACS Analysis for TRUS patients

Specimen collection：

Whole blood is collected in Vacutainer CPT （Becton-Dickinson） or 9 ml EDTA vials.

Isolation of peripheral mononuclear cells：

If CPT Vacutainer are used， we follow in principle the VACUTAINER CPT datasheet. Therefore the single steps are only described very shortly. For more information please always have a look into the datasheet！！ Changes are given in cursive letters.

Wash cell pellet a second time with PBS/2% horse serum and remove the supernatant with a Pasteur pipette and a waterpump as completely as possible.

Specimen Collection with VACUTAINER CPT

1. VACUTAINER CPT Tube with Sodium Citrate should be at room temperature.
　　2. Collect blood in the tube using the standard technique for VACUTAINER Brand Blood Collection Tubes and mix blood with sodium citrate solution immediately after collection.
　　3. Store tube upright at room temperature until centrifugation. Blood samples should be centrifuged within two hours of blood collection for best results. Remix the blood sample immediately prior to centrifugation by gently inverting the tube 8 to 10 times.
　　4. Centrifugation： room temperature / 8-10 min at 1500 x g （=3000 rpm， swingout rotor in the Sorvall table centrifuge， RIA Lab， program 4）.
　　5. Collect the whitish cell layer under the plasma layer （monocytes/macrophages and platelets） with a （2 ml plastic） Pasteur pipette. Transfer suspension to a 50 ml size conical centrifuge tube， fill up with cold PBS PBS containing 2% （horse） serum （or 1% BSA）） to 30 ml. Immediately collect the cells by centrifugation for 7 min at 244 x g （=1100 rpm， swingout rotor in the Sorvall table centrifuge， RIA Lab， program 4）. Remove supernatant by decanting to remove platelets.
　　6. Wash cell pellet a second time with cold PBS / 2% horse serum and remove the supernatant with a Pasteur pipette and a waterpump as completely as possible. Suspend the cells in PBS.