The MEC monolayers were washed three times with PBS 1× (Life Technologies) and gently dissociated from the insert filters followed by a 20-min fixation in paraformaldehyde (PFA) 4 % (w/v) (Sigma Aldrich, Saint-Louis, MO, USA) prior to immunocytochemistry. After three washes with PBS 1× (Life Technologies), the cells were pre-incubated for 30 min at room temperature (RT) with blocking buffer containing BSA 3 % (PAA Laboratories, Velizy-Villacoublay, France) in PBS 1×. The MEC monolayers were stained for 1 h in PBS 1× containing BSA 1 % (PAA Laboratories), with saponine 0.1 % (Sigma Aldrich) for membrane permeabilization, with a rabbit anti-occludin 1.5 μg/mL (Life Technologies). Cell nuclei were labeled with Hoechst 33342 1/1000 (Life Technologies) in co-incubation with a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Jackson Immunoresearch, West Grove, PA, USA). Cells were washed and mounted in Prolong Gold antifade mounting medium (Life Technologies). The mounted slides were observed with a Leica TCS SP2 confocal microscope (Leica Microsystems, Heidelberg, Germany). High-magnification images were acquired using a 63X HCX PL APO oil immersion objective and analyzed using the NIH ImageJ software (version 1.49o for Mac).
Pbs 1
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
PBS 1× is a sterile, buffered saline solution commonly used in various laboratory applications. It is a physiologically balanced salt solution that maintains the pH and osmotic pressure of biological samples. The core function of PBS 1× is to provide a stable and isotonic environment for the preservation and handling of cells, tissues, and biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde pfa, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Tcs sp2 confocal microscope, by Leica (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Rabbit anti occludin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 secondary antibody, by Jackson ImmunoResearch (1 mentions)
Phosphate buffered saline (pbs), by GE Healthcare (1 mentions)
Saponine, by Merck Group (1 mentions)
Clinac 2100 cd, by Agilent Technologies (1 mentions)
Rmp1 14, by Euromedex (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
Mem non essential amino acid solution, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Thp 1 cell line, by American Type Culture Collection (1 mentions)
Sodium pyruvate solution, by Thermo Fisher Scientific (1 mentions)
22 protocols using pbs 1
1
Immunostaining of Tight Junctions in MEC Monolayers
2
Preclinical Cancer Immunotherapy Protocol
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C57BL/6AnCrl and BALB/cAnCrl female mice were injected subcutaneously with 2.105 TC1, CT26, MC38, B16F10, or B16OVA cells in 100 µL of phosphate buffered saline (PBS 1×, Gibco, France) into the left flank. Tumor growth was monitored every 3 days with a caliper and groups were formed when tumor reached a mean diameter of 50–60 mm2. All CT procedures were conducted in the Pharmacy Department of the University Hospital of Besançon (France). Mice were administered a single dose of intraperitoneal cisplatin (5 mg/kg) and 5-fluorouracil (25 mg/kg). After 2 days, mice were anesthetized by injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and received single-fraction beam photon irradiation (8 Gy) using Clinac 2100 CD (Varian Medical System) with a maximal energy of 6 MV and in 2D with one beam. Mice from control group were injected with the solvent used to dilute the drug (the Pharmacy Department of the University Hospital of Besançon, France). Anti-mouse CTLA-4 antibody (9H10, Euromedex) and anti-PD-1 (RMP1-14, Euromedex) injections (200 µg/mouse two times a week for 2 weeks) started 2 days prior to CT and 3 days after RT, respectively. All experimental studies were approved by the local ethics committee in accordance with the European Union’s Directive 2010/63.
3
Differentiation of THP-1 Cells into Macrophage-like State
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The THP-1 cell line (from human acute monocytic leukemia) was obtained from ATCC and cultured in RPMI-1640 medium from Gibco. RPMI-1640 medium was supplemented with sodium pyruvate solution, L-glutamine, penicillin–streptomycin 1×, MEM non-essential amino acid solution, and 10% FBS, all purchased from Gibco. The macrophage-like state was obtained by treating THP-1 cells over 3 days with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA from Sigma-Aldrich). Afterward, adherent cells (THP-1 macrophage-like cells) were gently washed three times with PBS 1× from Gibco and rested with fresh RPMI-1640 medium for 5 days until use. Differentiation was verified, via the presence of CD14 marker in cells, using anti-human CD14 antibody conjugated to FITC (eBioscience, San Diego, CA, USA) in immunofluorescence assays (IFA). The antibody was incubated in paraformaldehyde (PFA)-fixed cells for 2 h at room temperature in the dark, and four washes were performed with PBS-Tween; then coverslips were mounted on slides using Vectashield (Vectorlabs, Burlingame, CA, USA). The slides were sealed and observed in an Axiovert200M microscope adapted to LSM 5 Pascal (Zeiss, Jena, Germany).
4
Evaluating CEC Migration Capacity
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The migration capacity of CECs was evaluated, by performing the scratch wound assay. CECs were initially cultured in 24 well plates (Costar Corning Life, Canton, MA, USA) with the cell culture medium, upon confluence (80–90%) reached. Then, the culture medium was totally removed, followed by brief washes with PBS 1× (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).
Finally, a gap occurred in the middle of the wells using 1 mL pipette tip, and the addition of 1 mL of α-MEM contained either 20% of CB-PL or CB-PPP, was performed. In positive and negative control groups, the same procedure as mentioned above was followed, and finally, either 1 mL of complete cell culture medium (positive control group) or 1 mL of α-MEM (negative control group) was added to the CECs cultures, respectively.
The migration of CECs was monitored until complete gap closure performance, and images were acquired at specific time points, using the inverted light microscope (Leica, DMII, Leica Microsystems, Wetzler, Germany) and processed with IC Capture v 2.4 software (Imaging Source, Bremen, Germany).
Finally, a gap occurred in the middle of the wells using 1 mL pipette tip, and the addition of 1 mL of α-MEM contained either 20% of CB-PL or CB-PPP, was performed. In positive and negative control groups, the same procedure as mentioned above was followed, and finally, either 1 mL of complete cell culture medium (positive control group) or 1 mL of α-MEM (negative control group) was added to the CECs cultures, respectively.
The migration of CECs was monitored until complete gap closure performance, and images were acquired at specific time points, using the inverted light microscope (Leica, DMII, Leica Microsystems, Wetzler, Germany) and processed with IC Capture v 2.4 software (Imaging Source, Bremen, Germany).
5
Isolation of Single Cell Populations
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Tissue samples were mechanically and enzymatically digested in CO2-independent medium (Gibco) containing 5% FBS (HyClone). Enzymatic digestion consisted of three rounds of 15 min of incubation with agitation at 37 °C, separated by pipetting, with 2 mg/ml collagenase I (C0130, Sigma), 2 mg/ml hyaluronidase (H3506, Sigma), and 25 µg/ml DNAse (Roche). The samples were filtered on a 40 µm cell strainer (Fischer Scientific) and were diluted in PBS 1× (Gibco) supplemented with EDTA 2 mM (Gibco) and 1% decomplemented human serum (BioWest). After two washes, cells were suspended in PBS before being stained for flow cytometry or flow sorting. PBMC were isolated from blood samples using FICOLL (GE Healthcare) gradient centrifugation.
6
Secretome Collection in Irradiated p53 Cell Lines
7
Multiparametric Flow Cytometry Analysis of Mouse T Cells
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Single-cell suspensions were prepared in FACS buffer: PBS 1× (Gibco) supplemented with 2% (vol/vol) FCS (Eurobio Scientific), 0.01% (vol/vol) sodium azide (Sigma-Aldrich). Cells were stained on ice in PBS 1× (Gibco) with Zombie Aqua Fixable Viability Kit (BioLegend). Cells were surface stained in FACS buffer with the following antibodies from eBioscience: FITC-labeled anti-mouse CD3ε (145-2C11), anti-CD16/32 (93); from BioLegend: BV785-labeled anti-mouse CD4 (RM4-5) and BV605-labeled anti-mouse CD8a (53-6.7). Cells were washed in FACS buffer. In mouse, CD4+ alpha beta T cells are CD3+ CD4+ CD8−.
After surface staining, cells were fixed using 4% (vol/vol) paraformaldehyde (PFA; Electron Microscopy Sciences) in PBS 1×. Cells were permeabilized in FACS buffer supplemented with 0.5% (wt/vol) saponin (Sigma-Aldrich). Intracellular staining was performed in the same permeabilization buffer with the following antibodies from eBioscience: APC/Cy7-labeled anti-IFN-gamma (XMG1.2); from BioLegend: PE-Cy7 anti-mouse IL-10 (JES5-16E3).
Flow cytometry was carried out by using LSR Fortessa X-20 (BD). Data were analyzed by using FlowJo software (BD Biosciences).
After surface staining, cells were fixed using 4% (vol/vol) paraformaldehyde (PFA; Electron Microscopy Sciences) in PBS 1×. Cells were permeabilized in FACS buffer supplemented with 0.5% (wt/vol) saponin (Sigma-Aldrich). Intracellular staining was performed in the same permeabilization buffer with the following antibodies from eBioscience: APC/Cy7-labeled anti-IFN-gamma (XMG1.2); from BioLegend: PE-Cy7 anti-mouse IL-10 (JES5-16E3).
Flow cytometry was carried out by using LSR Fortessa X-20 (BD). Data were analyzed by using FlowJo software (BD Biosciences).
8
Isolation of Murine White Adipose Tissue
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WAT of mice was rinsed in PBS 1× (Gibco) and cut into small pieces. Digestion buffer was added [1 ml/0.25 g of tissue; 2.5 mM glucose (Sigma-Aldrich, reference no. G8270), 2% fetal bovine serum (FBS), 200 μM adenosine (Sigma-Aldrich, reference no. 4036), and collagenase (1 mg/ml; Sigma-Aldrich, reference no. C2139) in Krebs-Ringer-Hepes (120 mM NaCl, 4,7 mM KCl, 2.2 mM CaCl2, 10 mM Hepes, 1.2 mM KH2PO4, and 1.2 mM MgSO4)] and incubated 1 hour at 37°C, on gentleMACS (Miltenyi). Following dissociation, WAT was filtered (150 μm; Partec GmbH), centrifuged 8 min at 150g, and resuspended in Hank’s balanced salt solution (HBSS1X) supplemented with 2% FBS and 200 μM adenosine. During centrifugation of digested WAT, the adipocytes formed a layer at the top of the liquid.
9
Eosinophil-Airway Smooth Muscle Cell Interaction
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Isolated eosinophils were separated into two parts: one part was used as control eosinophils before and 24 h after allergen challenge; another part of eosinophils was used for experiments with eosinophil integrins suppression peptide arginine-glycine-aspartate-serine (Arg-Gly-Asp-Ser, RGDS by Sigma Aldrich, Merck KGaA, St. Louis, MS, USA) (Figure 4 ). Respectively, the amount of eosinophils suspended in the serum-free growth medium was taken, and the solution of RGDS was added to the final concentration of 0.125 mg/mL. Freshly isolated eosinophils with RGDS were incubated for 1 h at 37 °C. After incubation eosinophils were centrifuged, the serum-supplemented growth medium was removed and resuspended in a fresh serum-free growth medium.
For ASM cells, cultivation dishes with approximately 2 × 105 cells were prepared, and combined cultures were made by adding suspension with 0.5 × 105 isolated viable eosinophils to the ASM cells. To observe and visualize the cell growth, an inverted microscope (CETI Inverso TC100; Medline Scientific, Oxford, UK) was used.
The combined cultures of ASM cells and eosinophils were incubated for 24 h. After incubation, eosinophils were washed out using warm PBS ×1 (GIBCO, Life Technologies) incubating co-cultures for 5 min at 36.6 °C and gently tapping on dish sides. ASM cells were then collected and lysed for gene expression analysis.
For ASM cells, cultivation dishes with approximately 2 × 105 cells were prepared, and combined cultures were made by adding suspension with 0.5 × 105 isolated viable eosinophils to the ASM cells. To observe and visualize the cell growth, an inverted microscope (CETI Inverso TC100; Medline Scientific, Oxford, UK) was used.
The combined cultures of ASM cells and eosinophils were incubated for 24 h. After incubation, eosinophils were washed out using warm PBS ×1 (GIBCO, Life Technologies) incubating co-cultures for 5 min at 36.6 °C and gently tapping on dish sides. ASM cells were then collected and lysed for gene expression analysis.
10
Cell Cycle Analysis by Flow Cytometry
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Cell cycle analysis was performed by measuring the DNA content of the treated and untreated cells using Propidium Iodide (PI) by flow cytometry. Briefly, cells were grown in 6-well plates (2 wells per condition) and incubated with AgNPs and G-AgNPs for 24 hours. Then, culture medium was collected, and the cells detached with trypsin/EDTA and centrifuged at 160 g for 3 minutes. The pellet was washed with PBS 1× (Gibco®, Gaithersburg, MD, USA) and fixed by slowly adding 3 mL of cold 70% (v/v) ethanol for 15 minutes at 4°C. Fixed cells were centrifuged and the pellet obtained was washed with cold PBS. Subsequently, the cells were stained with a solution containing Triton X-100 (1.1%, v/v), PI and RNase A (50 and 100 μg/mL, respectively) in PBS. The cell cycle analysis was made using the Cytomics FC500 flow cytometer (Beckman Coulter) and the CXP software. The percentage of cells in the sub-G1 phase of the cell cycle was calculated from the total 10,000 cells (100%) in the assay, and the percentage of cells in G0/G1, S and G2/M phases was calculated from the total cells excluding the sub-G1 cells. Three technical replicates were performed for each condition.
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