Alexa fluor 488 goat anti mouse antibody

The samples were stained for both vinculin and phalloidin and imaged using a Nikon D-Eclipse C1 Confocal Microscope—equipped with a Plan Apo VC 60XH objective- and an Olympus Fluoview FV10i Confocal Microscope—with an UPLSAPO 60XW objective. To do so, once the cells were fixed in 4% paraformaldehyde (Affymetrix) in PBS for 20 min at room temperature, samples were washed in PBS three times and permeabilized with 0.1% Triton X-100 (Calbiochem) in PBS at room temperature. Cells were washed another three times and blocked with 3% goat serum (Sigma) in 5% BSA/PBS solution for 4 h at room temperature. Afterwards, the devices were incubated overnight at 4 °C with mouse anti-human hVin-1 antibody (ab11194, Abcam) at 1:100 in 0.5% BSA/PBS. Then, after washing the samples five times with 0.5% BSA/PBS, incubation with Alexa Fluor^® 488 goat anti-mouse antibody (A11029, Molecular Probes) at 1:100 and the conjugated Alexa Fluor^® 594 phalloidin (A12381, Molecular Probes) at 1:200 was done for 3 h at room temperature in the dark. Finally, cells were washed three times with 0.5% BSA/PBS, two more times with PBS, and subsequently imaged.

Macrophage (0.8e6/per condition) were fixed for 20 minutes with 1 ml of fixation buffer (Invitrogen, 00822249) at room temperature (RT) in a FACS tube, washed twice with cold PBS, and then 1 mL permeablization buffer (Invitrogen, 00833356) was added for 15 minutes at RT. Cells were then centrifuged at 1400 rpm × 5 minutes at 4 °C and re-suspended in 500 ul blocking buffer containing 5% goat serum (gibco, 16210072). CFTR antibodies were used at a dilution of 1:100 (2784 CF3 Abcam, CFTR-594 and CFTR-570 from CFTR Antibody Distribution Program). Staining was done for 30 minutes at RT in the dark. Cells were washed twice with permeabilization buffer and re-supended in 100 uL Alexa Fluor488 Goat anti-mouse antibody (Molecular Probes, A31619) for 20 minutes at RT. Finally, cells were washed twice with permeabilization buffer and once with FACS buffer before FACS assay.

Cells were seeded at a concentration of 5×10³ or 1×10⁴ cells per 96-well and cultured in RPMI-1640 containing 0.5% FBS. Trypsinized cells were stained with trypan blue (TB) solution, and the non-stained viable cells were counted. For MTT assay, 20 µl of 5 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) was added to 5×10³ or 1×10⁴ cells per 96-well. After 4 hour-incubation at 37 °C, the culture soup was switched to 200 µl DMSO. The absorbance was measured at 595 nm. For BrdU assay, cells were grown with 10 µM bromodeoxyuridine (BrdU; Sigma-Aldrich Corp.) for 1 hr. Fixed cells by 4% PFA were probed with anti-BrdU antibody (Chemicon international Inc.). Fluorescence was detected by Alexa Fluor 488 goat anti-mouse antibody (Molecular Probe).

Anti-MHC antibody was obtained from the Developmental Studies Hybridoma Bank. Antibodies against GAPDH, phosphor-AKT (Ser-473), AKT, phosphor-ERK1/2 (Thr202/Tyr204) and ERK1/2 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase conjugated anti-mouse secondary antibodies were obtained from GE Healthcare Life Sciences (Little Chalfont, UK). Horseradish peroxidase conjugated anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Alexa Fluor 488 goat anti-mouse antibody and Hoechst 33342 were from Molecular Probes (Eugene, OR, USA). Formoterol hemifumarate and ICI-118,511 were from Bio-Techne Corporation (Minneapolis, MN, USA). Terbutaline hemisulfate was obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were from Sigma-Aldrich (St. Louis, MO, USA).

The ability of anti-capsule mouse polyclonal to bind to the surface of live meningococci was determined using flow cytometry analysis. Bacteria were grown in liquid GC medium to an OD600 of 0.5 and incubated with the anti-capsule mouse polyclonal serum diluted in PBS + 1% BSA (1:3200). After 1 hour, bacteria were washed with PBS + 1% BSA, incubated with a secondary Alexa Fluor 488 goat-anti-mouse antibody (1:1000, Molecular Probes) and fixed with 2% PFA. Cells were analyzed with a Canto II flow cytometer (Beckton-Dickinson) using FlowJo software.