Old PBS with 0.1 BSA and incubated for 20 min at 4uC with every single mAB (five mg/ml). Immediately after washing with cold PBS/BSA the cells had been analyzed by flow cytometry (FACSCalibur, CellQuest software program, BD Biosciences, Mountain View, CA) with data being collected on 104 viable cells. The following antibodies (mAB) had been used for immunofluorescence staining. Mouse IgG: CD14-PE (TUK4), CD3-PE (BW264/56), CD19-PE (LT19; Miltenyi Biotec, Bergisch-Gladbach, Germany), CD80-PE (B7-1), CD86-PE (B7-2), HLA-DR-FITC (eBioscience, San Diego, USA); and mouse-specific isotypes, IgG-PE (S43.10; Miltenyi Biotec, Bergisch-Gladbach, Germany) and IgG-FITC (679.1Mc7; Beckman Coulter, Fullerton, USA).Preparation of Entire Cell Extracts and Western Blot AnalysisMonocytes, DCs and macrophages had been harvested, washed once with ice-cold PBS, and lysed on ice in an appropriate quantity of lysis buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 1 mM EDTA, 0.1 Triton X-100, 2 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin and 97 mg/ml PMSF. Soon after 30 min incubation, lysates were centrifuged at 13.000 g and 4uC for 20 min along with the supernatant was recovered. The protein concentration was determined in line with Bradford [41]. Cell extract (30 mg) was separated on a 10 or 7.5 SDS polyacrylamide gel at one hundred V and was blotted onto a nitrocellulose membrane for 1 h at 300 mA making use of buffer composed of 25 mmol/L Tris-HCl, 86 mmol/L glycine, and 20 methanol. The antibodies utilized had been pATM Ser1981, p-cH2AX (Ser 139) (Millipore, Billerica MA, USA), pChk1 Ser317 (Bethyl, Montgomery TX, USA), pChk2 Thr68 ( Epitomics, Burlingame CA, USA), p53 (Dianova, Hamburg, Germany), pATR Ser428, Chk1, Chk2, XIAP, Cleaved Caspase-8, Cleaved Caspase-3, Cleaved caspase-7 (Cell signaling, Danvers, MA USA), XRCC1 (Abcam, Cambridge, UK), ligase IIIa, Poly(ADP-ribose) polymerase (PARP), FasL, Bcl2 (BD Biosciences), FasR, Bax and ERK2 (Santa Cruz Biotechnology, Heidelberg, Germany) as protein loading handle.Quantification of ApoptosisApoptosis was measured by subG1 assay. Right after therapy with TMZ, pretreated or not pretreated with O6-benzylguanine, monocytes, DCs and macrophages were washed in PBS, fixed in 70 ethanol for a minimum of 30 min at 220uC. DNA within the cells was stained with propidium iodide (16.five mg/ml) in PBS after RNase (0.03 mg/ml) digestion. For each and every sample 104 cells have been analyzed on a FACS Calibur (Becton Dickinson). The amount of apoptotic cells per sample was calculated using the personal computer plan Phleomycin Biological Activity WinMDI 2.PLoS 1 | plosone.orgMonocyte Response to TemozolomidePreparation of RNA, Semi-quantitative RT-PCR and Realtime RT-PCRTotal RNA was isolated from cells utilizing the RNA II Isolation Kit from Machery and Nagel. 1 mg RNA was transcribed into cDNA employing the Reverse-iT 1st Strand Synthesis Kit (ABgene, Surrey, UK). Primer sequences employed for PCR have been as follows: fasR (up, 59-AAGGGATTGGAATTGAGGAAGACTG-39; low, 59GTGGAATTGGCAAAAGAAGAAGACA-39) and gapdh (up, 59CCCCTCTGGAAAGCTGTGGCGTGAT-39; low, 59GGTGGAAGAGTGGGAGTTGCTGTTGA-39), which was utilised as loading handle. Real-time PCR was performed applying the SensiMix Plus SYBR Fluorescein Kit (Bioline) plus the MyIQ real-time PCR cycler (BioRad). Primer sequences used for real-time RT-PCR had been as follows: fasR (up, 59-TTATCT-GATGTTGACTTGAGTAA-39; low, 59-GGCTTCATTGACACCATT-39) and Actin (up, 59-TGGCATCCACGAAACTACC-39; low, 59-GTGTTGGCGTACAGGTCTT-39), which was employed as loading handle.AcknowledgmentsWe gratefully acknowledge Huong Becker for fantastic tec.