Cells remaining in the top compartment were eliminated having a cotton swab and membranes were mounted on a slip. To analyze 4T1 cell invasion, a modified Boyden chambers assay was achieved. After BAL collection, the right lung was clamped, harvested, and snap-frozen in liquid nitrogen. The remaining lung was insufflated with 4% paraformaldehyde and inlayed in paraffin for histologic analysis. For total protein extraction, lung cells were homogenized using a Mikro-Dismembrator device (Braun Biotech International, Melsungen, Germany). Crushed lungs were incubated over night at 4C in a solution comprising 2?M urea, 1?M NaCl, and 50?mM Tris (pH 7.5). Samples were then centrifuged for 15?minutes at 13?000for 10?moments at 4C, and pellets were cultured in DMEM supplemented with 10% FBS, 1% amino acids, 5?g/mL amphotericin B (Sigma), 0.5% gentamycin (Sigma), and 1% l-glutamine. Tumor cell colonies were visualized with Xenogen IVIS 200 after 4 to 6 6?days. Premetastatic niche characteristic analysis Collagen cross-linking was analyzed having a picro-red sirius staining. For this, slides were rehydrated and stained for 1?hour having a saturated aqueous remedy of picric acid containing 10% Red 80 (Sigma). Slides were washed twice having a 0.5% acetic acid solution and dehydrated in 100% ethanol. Then, after a bath of xylene, slides were mounted. Cross-linked collagen materials were visualized under a polarized light in optical microscopy. Quantification was performed using ImageJ software (5 blood vessels/slip, 5 sections/mouse). Results were indicated as the percentage between cross-linked collagen area Purpureaside C and the perimeter of the related blood vessel. Manifestation of lysyl oxidase was analyzed by reverse transcription-polymerase chain reaction using GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR kit (Perkin Elmer). Oligonucleotides were from Eurogentec (Seraing, Belgium) and were designed according to the sequence available in the GenBank: 5-TCCTCCAGACAGAAGCTTGCTT-3 (antisense) and 5-TGCCTGGCCAGTTCAGCATATA-3 (sense). The sequence specificity was verified using NCBI BLASTN system (http://www.ncbi.nlm.nih.gov/BLAST/). Retro-transcription was performed on 4-ng total RNA samples at 70C for 15?moments. Polymerase chain reaction steps consisted of 94C for 15?mere seconds, 60 for 20?mere seconds, and 72C for 10?mere seconds during 38 cycles Purpureaside C followed by 2?moments at 72C. Samples were migrated on a polyacrylamide gel and stained with GelStar (Biowhittaker, Indianapolis, IN, USA). Intensity of bands was quantified using Amount One software (Bio-Rad, Hercules, CA, USA). Analysis of gelatinase production and activity in the lungs was achieved by zymography as previously explained.39 Chemokine array and ELISA Chemokine array (R&D Systems, Wiesbaden, Germany) was performed according to the suppliers protocol. Membranes were 1st incubated with samples (pooled lung protein components, n?=?5) followed by a cocktail of biotin-labeled antibodies. Membranes were then incubated with streptavidin conjugated to horseradish peroxidase (HRP), and places were recognized with an ECL detection kit (Perkin Elmer). IL-16 detection in lungs, serum samples, and BALF and detection of s100A8, KC, and GM-CSF in 4T1-conditioned medium were performed with respective Mouse DuoSet ELISA packages (R&D Systems) according to the manufacturers protocol. European blotting Pooled protein components (n?=?10) were separated using a 16% polyacrylamide gel and transferred on a polyvinylidene fluoride membrane (Perkin Elmer), which was then blocked with a solution of PBS containing 10% dry milk and 0.1% Tween 20. A rabbit polyclonal anti-IL-16 (Santa Cruz, Santa Cruz, CA, USA) was applied on membranes over night at 4C. Membranes were washed and incubated CD209 having a swine anti-rabbit antibody conjugated to HRP (Dako, Purpureaside C Glostrup, Denmark) for 1?hour at Purpureaside C room temp. ECL detection kit and LAS 4000 (Fuji Picture Film Co., Tokyo, Japan) allowed the detection of interest bands. Blots were reprobed having a rabbit anti–actin.
Cells remaining in the top compartment were eliminated having a cotton swab and membranes were mounted on a slip
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