Fitc conjugated goat anti mouse igg

SH-SY5Y CTR, LMNA-KD cells and TICs were analyzed by indirect immunofluorescence. The cells were harvested, washed in cold 1X PBS and fixed (1 × 10⁶ cells/ml) in a solution containing cold acetone/methanol (1:4 v/v) in 50% 1X PBS. For each sample, 1 × 10⁶ cells were incubated with the human monoclonal antibody anti-N-Myc (Santa Cruz) in medium containing 10% FBS and 0.5% Tween 20 for 1 h at room temperature. After washing in PBS, the cells were incubated with FITC-conjugated goat anti-mouse IgG (BD, Pharmingen) in PBS for 50 min. After an additional wash in PBS, the samples were measured using a FACSCalibur cytofluorimeter (Becton Dickinson). Samples incubated with IgG isotype control antibody were used as negative controls, and NB LAN-5 cells were used as a positive control. The analysis was performed using the CellQuest software package. To determine the N-Myc immunofluorescence positivity we used the method of 2% of background.

Antigen-specific antibodies in the serum of one week after the third immunization were determined using ELISA. In short, 2-fold diluted serum was analyzed on 96-well plates (Nunclon, Roskilde, Denmark) coated with 0.1 μg NY-ESO-1 protein per well. NY-ESO-1-specific antibodies were probed with goat anti-mouse IgG, IgG2a or IgG1 conjugated with horseradish peroxidase (1:3000; ZSGB-BIO, Beijing, China). Finally, plates were read on an ELISA reader at a wavelength of 450 nm.
To determine the specificity of antibodies, serial dilutions of serum (1:400, 1:800, and 1:1600) were incubated with 3×10⁵ NY-ESO-1⁺ B16 cells for 1 h at 4°C after fixation and permeabilization. The cells were then washed in PBS, stained with FITC-conjugated goat anti-mouse IgG (BD Pharmingen, San Jose, CA) for 30 min at 4°C and analyzed using BD FACS Calibur flow cytometry (BD Pharmingen).

Anti-filaggrin antibody reactivity was determined by indirect immunofluorescence. Undiluted sera or sera diluted 1:10 in PBS from mice post exposed to the nickel nanomaterials, vehicles, and untreated mice were applied to rat oesophageal sections (Scimedx Corporation, Denville, NJ 07834, USA) for 1 hour at room temperature with humidity. The slides were then washed 3 times for 5 minutes in PBS. FITC conjugated goat anti-mouse IgG (BD Biosciences-Pharmingen) was applied for 1 hour at room temperature in a humid environment. The slides were again washed 3 times for 5 minutes in PBS then Dako mounting medium and cover slip placed on the slide. The samples were visualized using confocal microscopy (LSM Meta-510, Carl Zeiss, Axiovert, Germany).

Immunofluorescence staining of murine tumor cell lines was performed using culture supernatant of following murine hybridomas, kindly provided by Günter Hämmerling (DKFZ): S19.4 (anti-β₂m^b) [16 (link)], E3-25 (anti-H2-K^b) [17 (link)], B22.249 (anti-H2-D^b) [18 (link)]. Staining of IA^b molecules was performed using mononclonal antibody AF6-120 coupled to PE (BD) or to Alexa Fluor 647 (Biolegend, Fell, Germany), respectively. As controls, culture supernatants of hybridomas secreting isotype matched antibodies against irrelevant epitopes were included. Cells (2 x 10⁵) were incubated with 100 μl hybridoma supernatant for 1 hour at 4°C, followed by two washing steps and subsequent incubation with FITC-conjugated goat anti-mouse IgG (BD) as second antibody. Alternatively, cells were incubated with fluorochrome conjugated IA^b-specific antibody AF6-120 diluted in PBS containing 3% FCS. Finally, after staining with 7-AAD for live/dead discrimination, cells were analyzed with a FACSCalibur cytometer and data were evaluated with FlowJo software.

Momordicoside G (purity > 98% via HPLC) from M. charantia was purchased from Lianshuo Biotechnology Co., Ltd. (Shanghai, China). RPMI 1640 was obtained from Cellgro (Mediatech, United States). Fetal bovine serum (FBS) was obtained from Hangzhou Sijiqing Biological Engineering Materials Co. (China). Urethane, LPS, DAPI, CFSE, IL-10, AO, and Evans blue were purchased from Sigma Chemical Co. (St. Louis, MO, United States). MTT, DMSO were obtained from Life Technologies (United States). Antibodies used herein including anti-NLRP3, anti-arginase, anti-CD68, anti-iNOS, anti- LC3-B, anti-p62, β-actin, and FITC-conjugated goat anti-mouse IgG were obtained from BD Pharmingen. HRP-conjugated goat anti-mouse IgG polyclonal antibody, Annexin V-FITC Apoptosis kit, ROS, DCFH-DA and mouse quantitative ELISA kits (IL-6, IL-12, IL-10, and TGF-β1) were obtained from R&D Systems. Nitric oxide (NO) assay kit was obtained from Nanjing Jiancheng Bioengineering Institute. Standard rodent chow was purchased from Henan Provincial Medical Laboratory Animal Center (Zhengzhou, China), License No. SCXK (YU) 2015-0005, Certificate No. 41000100002406.

WJMSCs were analyzed for the expression of surface antigens and DNA content using flow cytometer (BD FACS Calibur; Becton Dickinson, NJ) in triplicates from three independent experiments. For phenotyping of surface antigens, WJMSCs were harvested using 0.25% Trypsin‐EDTA and fixed in 3.7% formaldehyde solution. The cells were then washed twice with DPBS and labelled (1 × 10⁵ cells per marker) with fluorescein isothiocyanate‐conjugated CD34 (BD Pharmingen, CA, FITC Mouse Anti‐Human CD34), CD45 (Santa Cruz Biotechnology, FITC Mouse Anti‐Human CD45), CD90 (BD Pharmingen, FITC Mouse Anti‐Human CD90) and unconjugated CD73 (Santa Cruz Biotechnologies, Mouse monoclonal), and CD105 (Santa Cruz Biotechnologies, Mouse monoclonal IgG_2a) for 30 min. Unconjugated primary antibodies were treated with secondary FITC‐conjugated goat anti‐mouse IgG (BD Pharmingen) for 30 min in the dark. For isotype matched negative control Mouse IgG1 (BD Pharmingen) was used. A total of 10,000 labeled cells per sample were acquired and results were analyzed using cell Quest Pro software (Becton Dickinson). For evaluating DNA content, a total of 1 × 10⁶ cells/ml were fixed in 70% ethanol at 4°C for 4 h. After washing cells twice with DPBS, they were stained with 10 µg/ml PI solution for 15 min. DNA content of each cell was measured and categorized as G₀/G₁, S, or G₂/M phase of the cell cycle.