Cells were fixed in BD CytofixTM fixation buffer (BD Biosciences) for 15 min at room temperature and then washed three times with BD perm/wash buffer (BD Biosciences) every 10 min. After a 30 min incubation with perm/wash buffer at room temperature, cells were stained with the following conjugated primary antibodies and incubated overnight at 4°C: OCT3/4-FITC (1:100; BD Pharmingen), NANOG-PE (1:100; BD Pharmingen), SOX2-PE (1:50; BD Biosciences), SSEA3-PE (1:100; BD Pharmingen), SSEA4-FITC (1:100; BD Biosciences), TRA-1-60-FITC (1:100; BD Pharmingen), and βIII-tubulin-FITC (1:100; BD Biosciences). Cells were imaged using fluorescence microscopy (Leica, Wetzlar, Germany).
Cytofixtm fixation buffer
Manufactured by BD
Sourced in United States
BD CytofixTM Fixation Buffer is a laboratory reagent used for the preparation of cell samples prior to flow cytometric analysis. It is designed to preserve the cellular structure and antigen expression of cells, ensuring accurate and reliable results during flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Perm wash buffer, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Nanog pe, by BD (1 mentions)
Ssea3 pe, by BD (1 mentions)
Ssea4 fitc, by BD (1 mentions)
Fluorescence microscopy, by Leica camera (1 mentions)
Sox2 pe, by BD (1 mentions)
Oleanolic acid, by Merck Group (1 mentions)
Vegf elisa kit, by Thermo Fisher Scientific (1 mentions)
Ursolic acid, by Merck Group (1 mentions)
Silver nitrate agno3, by Thermo Fisher Scientific (1 mentions)
Folin ciocalteu phenol reagent, by Merck Group (1 mentions)
Cell proliferation kit 2, by Merck Group (1 mentions)
Sodium tetrachloropalladate ii na2pdcl4, by Merck Group (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Phosflowtm perm buffer 3, by BD (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Cd45 percpcy5.5 clone 30 f11, by BioLegend (1 mentions)
6 protocols using cytofixtm fixation buffer
1
Comprehensive Stem Cell Characterization
2
Cytokine and Cell Proliferation Assays
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The VEGF ELISA kit, Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin-EDTA, phosphate—buffered saline (PBS), fetal bovine serum (FBS), antibiotics and PrestoBlue cell viability reagent were purchased from ThermoFisher Scientific (Johannesburg, South Africa). Sterile cell culture plates and flasks were obtained from Lasec SA (Pty) Ltd. (Midrand, South Africa). The BDTM Cytometric Bead Array (CBA) Human Inflammatory Cytokine kit, BD CytofixTM fixation buffer and the BD PhosflowTM permeation buffer were purchased from BD Biosciences (San Jose, CA, USA). The PGE2 ELISA kit was purchased from Biocom Biotech (Pty) Ltd. (Pretoria, South Africa). Sodium tetrachloroaurate (III) dehydrate (NaAuCl4) and silver nitrate (AgNO3) was procured from Alfa Aesar (Tewksbury, MA, USA). The Cell Proliferation Kit II (XTT) as well as all other chemicals and reagents, including actinomycin D (purity ≥ 95%), oleanolic acid (purity ≥ 97%), ursolic acid (purity ≥ 90%), human cyclooxygenase-2 enzyme, sodium tetrachloropalladate (II) (Na2PdCl4) and Folin—Ciocalteu phenol reagent were purchased from Sigma Chemicals Co. (St. Louis, MO, USA).
3
Visualization of SKIIP Localization in Stressed HeLa Cells
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HeLa cells were grown in 8-chamber glass slides. The expression of T7-epitope tagged SKIIP and SKIIP 3A was induced upon the addition of doxyciclin to the cell culture media at at 12.5 μg/ml concentration for 24 hours. Cells were then stressed with 100 mM NaCl for 60 minutes. After treatment, HeLa cells were fixed with BD CytofixTM Fixation Buffer and permeabilized with BD PhosflowTM Perm Buffer III (BD Biosciences) following the manufacturer’s indications. Fixed cells were then blocked with 3% BSA/TBS for 30 minutes. Rabbit anti T7 antibody (Novus) was incubated at a 1/500 dilution in blocking buffer overnight. The secondary anti-rabbit IgG-Alexa488 antibody (Life technologies) was incubated at a1/1000 dilution in blocking buffer for 1 hour in the presence of 8 μM Hoechst 33342 (Sigma) to stain the nuclei. Images were taken on an epifluorescense Nikon Eclipse Ti miscroscope.
4
Paw Cell Isolation and Characterization
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Paws were digested as described above, cell isolates were stained witn Ghost Dye TM Red 710 (Tonbo, San Diego, USA) according to the manufacturer´s recommendation and fixed with BD Cytofix TM Fixation buffer. Samples were stained after blocking of the FcR using mouse FcR blocking reagent (Miltenyi Biotec, Bergisch-Gladbach, Germany) with the following antibodies: CD45-PerCP/Cy5.5 (clone 30-F11, Biolegend), CD31-BV510 (clone 390, BD Bioscience), CD90. unmixing on a Cytek NL-3000 and data were analysed using SpectroFloR V3 (Scytek Biosciences, Freemont, USA) and FlowJo V10 (BD Biosciences).
5
Flow Cytometry Analysis of Th1/Th17 Cells
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At the end of the experiment, inguinal lymph nodes were collected from each mouse and ground using 70-μm nylon cell strainers (Falcon, Bedford, MA, United States) to isolate single cells. Subsequently, the cells were stained with a mouse Th1/Th17 phenotyping kit (BD Biosciences) according to the manufacturer’s instructions. Markers for Th1/Th17 were CD4 PerCP-Cy5.5-FITC-conjugated Th1 (IFN-γ), and CD4 PerCP-Cy5.5-PE-conjugated Th17 (IL-17A), respectively. Cells were stimulated for 4 h with phorbol myristate acetate and BD CytofixTM Fixation Buffer. Cell stimulation was terminated by fixing in 4% formyl saline. Fixed cells were stained in 0.1% BD Perm/WashTM Buffer for 30 min and finally analyzed on a FACSCalibur (BD Biosciences). The forward and side scatter gating method, a commonly used method in flow cytometer, was utilized for this analysis.
6
Immunolocalization of Neural Markers
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The immunolocalization of the target markers was performed by indirect staining with anti-human primary antibodies against TH, Neuronal Nuclei (NeuN) (Merck Millipore), Neurofilament (NEFM) (Merck Millipore), Dopamine Transporter (DAT) (Santa Cruz Biotechnology, Dallas, TX, USA), Musashi (Merck Millipore), Nestin (NES) (Merck Millipore), Neural Cell Adhesion Molecule (NCAM) (Merck Millipore). For myogenic differentiation, anti-human MYOG and MHC were used. Samples were fixed in BD CytofixTM Fixation Buffer (BD Biosciences) for 20 min at 4 °C, treated overnight at 4 °C with the primary antibody and incubated with FITC-conjugated secondary antibody (Merck Millipore) for 30 min, at RT. Then, samples were mounted with the mounting medium with DAPI (Merk) for nuclear counterstaining.
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