Syringe pump

Peripheral blood mononuclear cells were resuspended in Hank’s Buffered Salt Solution (HBSS) at a concentration of 5×10⁵ cells/ml and loaded into a syringe. HRMEC were grown in monolayers on glass slides coated with attachment factor and treated with drugs as outlined in the above sections. After treatment, slides were mounted in a rectangular parallel plate flow chamber with a silicon rubber gasket (GlycoTech; Gaithersburg, MD, USA). The chamber had a flow width of 1.00cm and a height of 0.0127cm. The chamber was connected to inlet and outlet syringes with Silastic™ tubing (GlycoTech) with an inner diameter of 1/16 inch. A syringe pump (World Precision Instruments; Sarasota, FL, USA) was used to pull PBMC across HRMEC monolayers at a rate of 150μl/min for 7min. Non-adherent cells were washed off the plate with HBSS at a flow rate of 300μl/min for 2min. Eight fields of view were captured using an IMT-2 inverted microscope (Olympus; Tokyo, Japan) and Q-Color3 digital camera (Olympus) at 20x magnification. Adherent cell counts were performed by masked observers using ImageJ (NIH; Bethesda, MD, USA) and averaged for each slide.

Stereotaxic injections were performed as previously described⁵¹ . Mice were anesthetized with isoflurane and placed into a stereotaxic apparatus (Kopf Instruments or Stoelting Instruments). After sterilization of the incision site with ethanol and Betadine, the skull was exposed via a small incision. Small burr holes were drilled above the microinjection target sites. A 2μl Neuros Hamilton Syringe with a 30-gauge needle was used to microinject virus into the target sites at a rate of 100 nl/min using a syringe pump (World Precision Instruments). The needle was slowly withdrawn 5 minutes following the completion of the injection to reduce backflow of the virus. Target coordinates relative to Bregma (in mm) and viral volumes injected were: ARC (AP: −1.50 to −1.55, ML: ± 0.30, DV: −5.85, 700 nl per side). AcbSh (AP: 1.00, ML: ±1.70 to 2.00, DV: −4.40 to −4.60, 400 nl per side). Following surgery, the incision was closed using surgical sutures. In some cases, viral injections were performed using a pulled glass pipette, as previously described^{6 (link)}. In these cases, the following coordinates were used. ARC coordinates: AP −1.45mm, ML +/− 0.25mm, DV −5.75mm and −5.65mm (300 nl per side). VTA coordinates: AP −3.05mm, ML +/− 0.35mm, DV −4.45mm and −4.25mm (150 nl unilaterally). DMH coordinates: AP −1.80mm, ML +/− 0.30mm, DV −5.2mm.