Recombinant timp1

Phorbal 12-myristate 13-acetate (PMA) and Ionomycin were purchased from Sigma-Aldrich. PMA was reconstituted to 10mg/ml stocks in DMSO and was further diluted to a working concentration of 40ng/μl in AIMV medium. Imiquimod, a TLR7 agonist, was purchased from LKT Laboratories (St Paul, MN) and reconstituted to 1mg/ml in DMSO. Recombinant TIMP1, TIMP2, TIMP3, and TIMP4 were purchased from R&D Systems and were reconstituted to working concentrations in AIMV medium. The protease inhibitors GM6001 and TAPI-0 were purchased from Calbiochem and reconstituted to 10mM and 1mg/ml stock, respectively, in DMSO.
The monoclonal rat anti-hCD200 antibodies 1B9 and 3G7, and the polyclonal rabbit serum against the extracellular region of CD200 (CD200v+c), were described previously [2 (link)]. A polyclonal rabbit serum against the human CD200 receptor (CD200R1) was generated by immunization of rabbits with a fusion protein containing the extracellular region of human CD200R1 with a his-tag at the N-terminal.
Antibodies against CD19 and CD62L used in FACS analyses were purchased from Biolegend. The apoptosis detection kit for staining of Annexin V and 7AAD was purchased from BD Biosciences. The Pan-Cadherin antibody, used as a plasma membrane marker for loading controls in Western blots, was purchased from Abcam.

HUVEC cells were plated in 24-well plates (1.5 × 10⁵ cells per well) two days before the scratch test. Confluent cells were starved for 3 h, and a scratch wound was made using a sterile pipette tip. Cells were then washed to remove any loosely held cells, and the CM was added in the presence or not of 50ng/mL of recombinant TIMP-1 (R&D Systems) or anti-hTIMP-1 (1 µg/mL) (or goat IgG as control). The open wound was monitored at the microscope (Leica Time Lapse AF6000LX workstation (Leica Microsystems, Milano, Italy) interfaced with the Leica Application Suite (LAS) software (AF6000) for 12 h. Analysis was performed using ImageJ software to quantify the extent of the migratory front.