The plates were washed with PBS and blocked with 1% polyvinylpyrr

The plates were washed with PBS and blocked with 1% polyvinylpyrrolidone (Sigma, Munich, Germany) at room temperature for 1 h and then washed https://www.selleckchem.com/products/Nolvadex.html extensively with PBS at 37°C for 40 min. A total of 2.5 × 105 neutrophils in 500 μL of DPBS were pretreated with rmTNF (50 ng/mL at 37°C for 15 min) and then added to the wells for 40 min. Plates were then washed gently three times with prewarmed PBS and the remaining adherent cells were quantified by counting three microscopic fields at a 40× magnification. RNA was prepared as described [44]. Briefly, murine PMNs were isolated and RNA was immediately prepared with TriFast (Peqlab, Erlangen, Germany). Reverse transcription

was performed on 1 μg of RNA using random hexamers reverse transcriptase. A total of 200 nM of the resulting cDNA was then subjected to 40 cycles of PCR in a 25 μL reaction mixtures in a BioRad cell cycler (BioRad, Munich, Germany). The PCR products were subjected to agarose gel analysis; m24p3R fw: GGC GAT TTC TAC AGG GAA TGA rv: CTA TCA GCC ACC GTG CAG ACT; mMegalin fw: TGC HDAC assay ACG GAG GAA GTT GCT ATT rv: TCC ACT GTA GCC GCT AGA ACA. Rabbit polyclonal sera were raised against 24p3R. The sequences of the synthetic peptid used and

the location within the primary amino acid sequence was CDHVPLLATPNPAL (anti-24p3R: 507–520). Crude serum was affinity purified. Antibody production and affinity purification were performed by Eurogentec (LIEGE Science Park, Belgium). Protein extracts were prepared from freshly isolated human PMNs using cytoplasmic

lysis buffer (50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% NP-40 with protease inhibitors). Ten micrograms of protein were resolved by SDS-PAGE (BioRad) and transferred to nitrocellulose membranes (Amersham Hybond-P; GE Healthcare, Buckinghamshire, UK). Membranes were blocked with 4% blocking milk/TBS/Tween and incubated with Abs against anti-human 24p3R (Eurogentec) and antiactin (Sigma). Oxyblots were performed using the ADP ribosylation factor SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Vienna, Austria) according to the manufacturer’s instructions. Freshly isolated murine blood was drawn by retroorbital puncture. Samples were stained with anti-mouse CD11b Alexa Fluor 488 (M1/70, Biolegend, Uithoorn, the Netherlands), anti-mouse Ly-6G/Ly-6C PerCP (RB6–8C5, Biolegend), anti-mouse Ly-6G FITC (1A8, Biolegend), anti-mouse CD182 PerCP/Cy5.5 (TG11/CXCR2, Biolegend), anti-mouse CD184 Alexa Fluor 647 (TG12/CXCR4, Biolegend), anti-mouse CD51-PE (RMV-7, Biolegend), anti-mouse CD62L Alexa Fluor 647 (MEL-14, Biolegend), or appropriate isotype IgGs. Cells were measured with BD FACS Calibur flow cytometer (BD Bioscience, Heidelberg, Germany) and analyzed with Kaluza Software (Beckman-Coulter, Vienna, Austria).

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