To establish proof of principle for

this assay, we analyzed fresh blood samples from a population of individuals who are at high risk for having a germline MMR mutation Methods Materials Human colorectal cancer cell lines (SW480, LoVo, HCT116), culture media (RPMI-1640, MEM, F-12 K), Fetal Bovine Serum (FBS), Trypsin/EDTA and antibiotics were purchased from American Type Culture Collection (ATCC). Antibodies were Necrostatin-1 purchase from the commercial sources indicated (Table 1). M-PER mammalian protein extraction reagent was from Pierce Biotechnology. Anti-mouse-IgG-HRP conjugated detection antibody, protease inhibitor cocktail, PMSF, 2-mercaptoethanol, PHA, penicillin, and streptomycin were from Sigma-Aldrich. Lymphoprep was from Axis-Shield. Human IL-2 was a gift from Dr. Martin Cannon, University of Arkansas for Medical Sciences,

Little Rock, AR. Table 1 Commercially available monoclonal and polyclonal antibodies used VX-680 manufacturer for detection of MLH1 and MSH2 proteins on western blots. No. Names Catalog Number Company Monoclonal Antibodies       1 Anti-MSH2(Ab-2) mouse mAb(FE11) NA27 EMD Calbiochem, Gibbstown, NJ 2 MLH1 554073 BD Pharmingen, San Diego, CA 3 Anti-MSH2(Ab-1)mouse mAb(GB12) NA26T Calbiochem, San Diego, CA 4 Anti-MLH1(Ab-1)mouse mAb(14) NA28 Calbiochem, San Diego, CA 5 MLH1 Sc-56159 Santa Cruz, Santa Cruz, CA 6 MLH1 Sc-56161 Santa Cruz, Santa Cruz, CA 7 MSH2 Sc-56163 Santa Cruz, Santa Cruz, CA 8 MSH2 556349 BD Pharmingen, San Diego, CA Polyclonal Antibodies       1 MLH1(N-20) Sc-581 Santa Cruz, Santa Florfenicol Cruz, CA 2 MSH2 (N-20) Sc-494

Santa Cruz, Santa Cruz, CA 3 Anti-MSH2 (Ab-3) Pc57 Calbiochem, San Diego, CA 4 Anti-MLH1 (Ab-2) Pc56 Calbiochem, San Diego, CA 5 Rabbit anti-MSH2 A300-020A Bethyl Labs, Montgomery, TX 6 MLH1 2549.00.02 Sdix, Newark, DE Isolation of Lymphocytes After IRB approval and signed informed consent, venous blood was collected from patients using EDTA-containing vacutainer tubes. Samples were collected from individuals undergoing genetic counseling for hereditary colon cancer in the Familial Cancer Clinic at the Helen F Graham Cancer MRT67307 Center, Christiana Care Health System (Newark DE). Samples were de-identified and processed within 24 hours to isolate lymphocytes. Lymphocytes were separated by density gradient centrifugation using Lymphoprep. Briefly, blood samples were diluted 2-fold with PBS, pH 7.4. An aliquot of 20 ml diluted blood was layered over 15 ml of Lymphoprep in 50 ml Falcon centrifuge tubes and centrifuged at 1000 g for 20 min at room temperature in a Sorvall RC 6 Plus centrifuge using an SH 3000 swinging bucket rotor. Lymphocytes were harvested from the buffy coat; monocytes from the plasma layer. Lymphocytes were diluted 3-fold with PBS (pH 7.