AIM assays were performed as previously described 43 (link). Briefly, cryopreserved PBMC were thawed, washed, resuspended in R10, and rested for 3 hours at 37°C. Following the 3-hour rest interval, the appropriate number of cells were transferred to a 48-well plate and subsequently treated with CD40 blocking antibody (Miltenyi Biotec, cat. no. 130–094-133) for a final concentration of 0.5ug/mL for 15 minutes at 37°C. Cells were incubated in the presence or absence of our 10-LRA panel, as previously described. After an 18hr incubation, cells were harvested and stained for 50 minutes at 4°C with the surface staining monoclonal antibodies (mAb); (PD-L1-PE/Cy7 (Biolegend, cat. no. 329717), CD40L-PE (BD Biosciences [BD], cat. no. 561720), OX40-APC (BD cat. no. 563473), CD69-BV650 (Biolegend cat. no. 310933), CD3-BV605 (Biolegend cat. no. 317322), CD4-BV421 (BD cat. no. 562424), CD8-PerCp-Cy5.5 (BD cat. no. 560662), CD25- BUV395 (BD cat. no. 564034) and LIVE/DEAD Near-IR stain (ThermoFisher cat. no. L34975), washed, fixed and permeabilized (BD cytofix fixation buffer, cat. no. 554655), and then stained for 30 minutes at 4°C with intracellular mAb Acetyl-histone H3-Alexa Fluor 488 (Cell Signaling Technology, cat. no. 9683S) in 1x perm/wash buffer (BD, cat. no. 554723). The cells were washed and fixed (BD cytofix fixation buffer, cat. no. 554655) prior to flow cytometry analysis.
Cytofix fixation buffer
Manufactured by BD
Sourced in United States, Italy, United Kingdom, Uruguay
BD Cytofix Fixation Buffer is a laboratory reagent designed to fix and preserve cellular samples for further analysis. It is a formaldehyde-based solution that crosslinks proteins within cells, maintaining their structural integrity and antigen presentation. The product is intended for use in flow cytometry and other cell-based applications requiring fixed samples.
Automatically generated - may contain errors
Lab products found in correlation
Phosflow perm buffer 3, by BD (42 mentions)
Perm wash buffer, by BD (36 mentions)
Perm buffer 3, by BD (17 mentions)
Facscanto 2, by BD (10 mentions)
Lsrfortessa, by BD (10 mentions)
Facscalibur, by BD (9 mentions)
Lsrii flow cytometer, by BD (9 mentions)
Ionomycin, by Merck Group (9 mentions)
Stain buffer, by BD (9 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (8 mentions)
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
Facsdiva, by BD (5 mentions)
Triton x 100, by Merck Group (5 mentions)
Facscanto 2 flow cytometer, by BD (5 mentions)
Lsrfortessa flow cytometer, by BD (5 mentions)
Facsdiva software, by BD (4 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (4 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (4 mentions)
Brefeldin a, by BD (4 mentions)
Fortessa flow cytometer, by BD (4 mentions)
221 protocols using cytofix fixation buffer
1
Investigating Immune Checkpoint Modulation in PBMCs
2
Immunofluorescence Staining of hNPC-MCs
3
Tissue Preservation and Cryosectioning for Fluorescent Imaging
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Tumor tissue with the overlying skin was collected, fixed with BD Cytofix fixation buffer (554655; BD) overnight, then preserved with 30% sucrose (S8501; Sigma-Aldrich) overnight prior to embedding and freezing in O.C.T. (Tissue-Tek). Then 8-μm-thick frozen sections were cut in a cryostat (OTF5000; Bright), fixed with BD Cytofix fixation buffer, and then stained with a far red DNA dye Draq5 (1:1,000, 424101; BioLegend). Images were taken using a Zeiss LSM 880 microscope (Zeiss) and analyzed with ImageJ.
4
Intracellular Phospho-NF-κB Analysis
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Cells were fixed with Cytofix Fixation Buffer (BD Biosciences, San Jose, CA), permeablized with Phosflow Perm Buffer III (BD Biosciences), then stained for 30min with optimal concentrations of PE-conjugated antibodies to p-NF-κB (Cell Signaling, Danvers, MA) or irrelevant antibodies of the same isotype and flourochrome. After staining, the cells were washed, fixed in Cytofix Fixation Buffer (BD Biosciences), and analyzed by flow cytometry. Data were analyzed with FlowJo software version 10.0.
5
Intracellular Phospho-NF-κB Analysis
6
Comprehensive Stem Cell Characterization
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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).
7
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).
8
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.
9
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).
10
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.
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