Bacterial abundance (BA) was determined by flow cytometry technique (FACSCanto II, Becton Dickinson Biosciences, Oxford, UK) fixing 1.5 mL of sampling water with particle-free 20% (w/v) paraformaldehyde, 1% final concentration followed by liquid nitrogen frozen and stored at −80°C [68] , [69] (link). Before being analysed, the samples were thawed and stained with Syber Green I DNA (Sigma-Aldrich) 1∶5000 final dilution of initial stock [69] (link), [70] . Yellow-green 1-µm beads at a standard concentration (Fluoresbrite Microparticles, Polysciences, Warrington, PA, USA) were added in order to determine absolute cell concentrations [69] (link), [71] (link).
Fluoresbrite microparticles
Manufactured by Polysciences
Sourced in United States
Fluoresbrite Microparticles are fluorescent polymer microspheres designed for use in various applications including flow cytometry, microscopy, and immunoassays. These particles are available in a range of sizes and fluorescent dye options to meet the needs of different research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Syber green 1 dna, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Cytation 1 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
Polylink protein coupling kit, by Polysciences (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Facsort, by BD (1 mentions)
Anti ng2, by Merck Group (1 mentions)
Anti pdgfrα, by Neuromics (1 mentions)
Anti olig2, by Merck Group (1 mentions)
Lysolecithin lpc, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
6 protocols using fluoresbrite microparticles
1
Bacterial Abundance Quantification by Flow Cytometry
2
Macrophage Phagocytic Assay with Oxalate
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BMDMs were seeded in a 6-well plate (106 cells/well) and allowed to rest for 24 hrs at 37°C in a CO2 incubator. Cells were then exposed to calcium oxalate (CaOx; 50 μM) or sodium oxalate (NaOx; 50 μM) for 72 hrs. Subsequently, macrophages were washed and fluorescent latex beads (Cat. #15702-10 Fluoresbrite® Microparticles, Polysciences) were added to the wells (100 beads/cell reconstituted in 1x PBS) (38 (link), 39 (link)). Afterwards, cells were kept at 37°C for 30 minutes while shaking. Cells were washed twice with 1x PBS and visualized using a Cytation 1 Cell Imaging Multimode reader (BioTek) at 488/510 nm.
3
Immunodetection of Scrub Typhus Antigens
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Both preimmune mouse serum and anti-Sca polyclonal mouse serum (produced from Balb/c mice immunized with purified Sca proteins; Cosmogenetech, Seoul, South Korea) were used for the experiments. Human sera were prepared from scrub typhus patients following institutional review board approval. Horseradish peroxidase (HRP)-conjugated anti-mouse or anti-human IgG secondary antibodies (Santa Cruz Biotech Inc., Santa Cruz, CA) were used for immunoblotting [32 (link)]. The Alexa Fluor 488- or Alexa Fluor 594-conjugated anti-mouse, and-human antibodies used in the immunofluorescence assays were purchased from Molecular Probes (Invitrogen). For the beadbinding assay, Fluoresbrite microparticles (1 μm; Polyscience Inc., Warrington, PA) containing rhodamine were conjugated to GST or GST-ScaA proteins by using a PolyLink protein coupling kit (Polyscience Inc.) in accordance with the manufacturer’s instructions.
4
Quantifying Bacterioplankton Biomass in Seawater
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Seawater samples were fixed with 2% (w/v) paraformaldehyde for 1 h at room temperature (RT). Bacterial cells were stained with SYBR Green I DNA dye25 (link) and counted by flow cytometry (FACSort flow cytometer, Becton Dickinson) using the CellQuest software. Bacterioplankton concentrations were determined using 0.5 μm yellow-green micro-spheres (Fluoresbrite Microparticles, Polyscience) as an internal standard26 (link). To convert bacterioplankton concentrations (N, cells l−1) into biomass (B) we used a mean cellular biomass (Bc) of 11.5 fg carbon or protein cell−1 27 (link): under the assumption of equal cell carbon and protein contents28 .
5
Immunohistochemical Analysis of Oligodendrocyte Development
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Lysolecithin (LPC), Paraformaldehyde (PFA), Triton X-100 and Hoechst 33,342 were obtained from Sigma (St. Louis, MO, USA). Anti-MBP antibody was a generous gift from Dr. Anthony Campagnoni (Mental Retardation Research Center, University of California, Los Angeles, CA, USA). Anti-NG2, anti-Olig2 and RIP antibodies were obtained from Millipore (Billerica, MA, USA). Anti-PDGFrα, anti-glial fibrillary acidic protein (GFAP) and anti-Nestin antibodies were obtained from Neuromics (Edina, MN, USA). Anti-Ed1 and anti-Iba-1 antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-iNOS antibody was purchased from Becton Dickinson (Franklyn Lakes, NJ, USA) and biotinylated Griffonia Simplicifolia was obtained from Vector Laboratories (Burlingame, CA, USA). Secondary antibodies were obtained from Jackson Immuno-Research Co. Laboratories (West Grove, PA, USA). All other chemicals used were of analytical grade of the highest available purity. Anti-Gal-1 antibody was prepared in G.A.R's laboratory and used as described (Dettin et al., 2003) . Fluoresbrite Microparticles were obtained from Polysciences Inc. (Warrington, PA, USA).
6
Evaluating Epithelial Barrier Function
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Electrical Resistance (TEER). Monolayers were gently washed with pre-warmed HBSS and measured at 37°C using an Ag/AgCl electrode. TEER values are depicted in unit area resistance (Ω cm 2 ) = resistance (Ω) x effective membrane area (cm 2 ).
To detect functional changes in mono-versus co-cultures, transport efficiency of monolayers was analyzed using fluorescent microspheres. Fluoresbrite® Microparticles (0,46 µm; Polysciences, Inc.) diluted in cell culture medium were introduced into the apical chamber of transwells. The basolateral solution was sampled at several time points and analyzed using an ELISA reader. Percentage of translocation was calculated using the donor solution for calibration. After incubating the co-culture in the microsphere solution for 250 minutes, monolayers were analyzed microscopically as described earlier.
To detect functional changes in mono-versus co-cultures, transport efficiency of monolayers was analyzed using fluorescent microspheres. Fluoresbrite® Microparticles (0,46 µm; Polysciences, Inc.) diluted in cell culture medium were introduced into the apical chamber of transwells. The basolateral solution was sampled at several time points and analyzed using an ELISA reader. Percentage of translocation was calculated using the donor solution for calibration. After incubating the co-culture in the microsphere solution for 250 minutes, monolayers were analyzed microscopically as described earlier.
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