For ex vivo cytokine analysis, cells were first treated with a 1× cell activation cocktail of PMA/ionomycin/Brefeldin A (Biolegend for 5 h in complete RPMI media. 1 × 106 cells from single cell suspensions were stained with anti-CD16/CD32 Fc block (1:200, BD Biosciences) and fixable viability 700 dye (1:10,000, BD Biosciences) in 1× PBS for 15 min. at 37 C prior to surface and intracellular staining with primary antibodies. Surface staining commenced in 200 μl FACS buffer supplemented with a 1:4 dilution of Brilliant Violet stain buffer (BD Biosciences) and fluorescently conjugated antibodies from the table below for 30 min. at 4 C in the dark; all are mouse reactive antibodies unless otherwise indicated. Following surface staining cells were washed and fixed in either 2% PFA or Fix/Perm buffer (eBioscience) for 20 min. at 4 °C for intracellular stain. Fix/Perm buffer was washed with 1× perm wash buffer (eBioscience) and intracellular proteins were stained with fluorescently conjugated ICS antibodies in perm wash buffer for 30 min at 4 °C in the dark. Cells were washed and resuspended in 1× PBS prior to acquisition on a BD Fortessa or LSRII flow cytometer (BD Biosciences). Please refer to Supplementary Table 1 for a complete list of antibodies used.
Fix perm buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fix/Perm buffer is a laboratory reagent used for the fixation and permeabilization of cells. It is designed to enable the access and detection of intracellular targets during flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Permeabilization buffer, by Thermo Fisher Scientific (20 mentions)
Fortessa, by BD (6 mentions)
Iridium intercalator solution, by Standard BioTools (6 mentions)
Ddh2o, by Standard BioTools (6 mentions)
Ionomycin, by Merck Group (5 mentions)
Facscanto 2, by BD (5 mentions)
Facscalibur, by BD (5 mentions)
Golgiplug, by BD (5 mentions)
Perm buffer, by Thermo Fisher Scientific (5 mentions)
Lsrii flow cytometer, by BD (4 mentions)
Cytoflex, by Beckman Coulter (3 mentions)
Golgistop, by BD (3 mentions)
Cell stimulation cocktail plus protein transport inhibitor, by Thermo Fisher Scientific (3 mentions)
Lsrfortessa, by BD (3 mentions)
Facs lsr 2, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Leukocyte activation cocktail, by BD (3 mentions)
Fc500, by Beckman Coulter (2 mentions)
Fc receptor blocking agent, by Miltenyi Biotec (2 mentions)
112 protocols using fix perm buffer
1
Ex Vivo Cytokine Analysis Protocol
2
Isolation and Analysis of Tumor-Infiltrating Lymphocytes from Ovarian Tissues
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The TILs were harvested from fresh surgical resected ovarian cancer and benign ovarian cyst tissues according to the previously described methods [13 (link)]. Mouse anti-human CD45-PerCP, mouse anti-human CD4-APC-Cy7, mouse anti-human CD8α-PE-Cy7, mouse anti-human CD56-APC, mouse anti-human T-bet-PE antibodies, fluorescence labeled mouse or rat IgG Isotypes, and the FixPerm buffer were all purchased from eBioscience (San Diego, CA). The TILs were isolated from ovarian cancer and benign ovarian cyst by using the standard ficoll-hypaque density gradient centrifugation. For intracellular staining of T-bet, the TILs were surface stained, then permeabilized with the eBioscience FixPerm buffer. Subsequently, the cells were washed and stained with mouse anti-human T-bet-PE or mouse IgG1-PE Isotype Control. Flow cytometric analysis was performed using BD Canto II cytometer (BD Biosciences, San Jose, CA, USA), and the data was analyzed using FlowJo 10.0.6 software.
3
Quantifying Plin2 Expression in Immune Cells
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To assess Plin2 basal expression, PBMCs from obese IR, NAFLD subjects and healthy controls were fixed and permeabilized in FIX/PERM buffer (eBioscience San Diego, CA), and stained for Plin2 using AlexaFluor 488 as secondary antibody. Results were expressed as MFI. To identify monocyte subpopulation, CD16-PeCy7 and CD14-ECD were used. PBMCs and HepG2 stimulated with HSPs (50–800 pg mL−1), were fixed and permeabilized in FIX/PERM buffer (eBioscience San Diego, CA) and stained for Plin2 using AlexaFluor 488 as secondary antibody. Flow cytometric analysis was conducted with FC 500 (Beckman Coulter, Brea, CA) and the data analyzed with Kaluza software (Beckman Coulter, Brea, CA).
4
Flow Cytometric Identification of Osteoblast and Osteoclast Precursors
5
Multiparametric Immunofluorescence Profiling of PBMCs
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Immunofluorescence staining was performed on PBMCs. Briefly, cells were incubated with Fc receptor blocking agent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with monoclonal antibodies for 20 min at 4 °C in a darkened room. The following monoclonal antibodies were used (all from BioLegend, San Diego, CA, USA): FITC-CD3 clone OKT3, PerCP/Cy5.5-CD14 clone HCD14, APC-CD69 clone FN50, PE-CD142 clone NY2. After fixation of stained cells using Fix/Perm buffer (Thermo Fisher Scientific, Waltham, MA, USA), the suspension of fixed cells was immobilized onto glass slides by cytospin. Nuclei were counter stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (DOJINDO, Kumamoto, Japan) in water, and whole sections were mounted in ProLong Diamond (Thermo Fisher Scientific, Waltham, MA, USA). Slides were observed with a confocal fluorescence microscope (FV3000, Olympus, Tokyo, Japan).
6
Multicolor Flow Cytometry Immune Profiling
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Single cell suspensions were made by passing spleens through a 70 μm cell strainer, washed three times with FACS buffer (0.01 M azide, 2% FBS, PBS), incubated with blocking antibody for 10 min at 4°C, then labeled with surface marker-specific fluorochrome-labeled antibodies (Supplementary Table 1) for an additional 20 min at 4°C and unbound Ab was washed away with FACS buffer. Cells that were probed for intracellular markers were permeabilized (Fix/Perm Buffer; ThermoFisher Scientific, Waltham, MA, USA) for 30 min, washed again with Perm buffer, incubated with blocking antibody in the same buffer for 10 min, and then labeled with marker-specific or isotype control fluorochrome-labeled antibodies for an additional 20 min. Stained cells were washed twice with Perm buffer and once with FACS buffer, and analyzed on a CytoFLEX flow cytometer (Beckman Coulter, Mississauga, ON). The data was processed using FlowJo software (Tree Star Inc., Ashland, OR). Supplementary Figure 2 shows gating scheme for the identifications of various immune cells.
7
Intracellular Protein Analysis Protocol
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For analysis of intracellular proteins, cells were fixed and permeabilized with Fix/Perm buffer (ThermoFisher Scientific, Waltham, MA, USA) and then incubated with primary monoclonal antibody anti-ARG1 (C-2) (Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-mouse Alexa Fluor®-488 (ThermoFisher Scientific, Waltham, MA, USA) secondary monoclonal antibody.
8
Immunolocalization of human IgG in hAECs
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Primary human alveolar epithelial cells (hAECs) obtained from Generon were maintained in Endothelial cell growth medium‐2 (Promocell) at 37 °C in a 5% CO2 humidified atmosphere. hAECs were seeded at a density of 50 000 cells per well in a 96‐well tissue culture plate. Cells were then treated with either human IgG or D‐PCF overnight. The following day, cells were harvested for confocal imaging and flow cytometry analysis.
For confocal imaging, hAECs were washed twice with PBS and fixed with Fix/Perm buffer (ThermoFisher Scientific) according to the manufacturer's instructions. Cells were then permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich) for 10 min and stained with phalloidin, DAPI and anti‐human IgG‐FITC (Biolegend). After washing and resuspension in PBS, cells were imaged using a confocal microscope (Nikon A1R).
For flow cytometry analysis, human IgG or D‐PCF‐treated cells were washed twice with PBS and stained with eFluor870 fixable viability dye (ThermoFisher Scientific) for 20 min at 4 °C. The cells were then fixed with Fix/Perm buffer for 20 min at 4 °C, permeabilized with 0.1% Triton X‐100 for 10 min and stained with anti‐human IgG‐FITC for 1 h at 4 °C. Flow cytometry acquisition was performed using the Cytoflex (Beckman Coulter) and analysed using FlowJo V10.
For confocal imaging, hAECs were washed twice with PBS and fixed with Fix/Perm buffer (ThermoFisher Scientific) according to the manufacturer's instructions. Cells were then permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich) for 10 min and stained with phalloidin, DAPI and anti‐human IgG‐FITC (Biolegend). After washing and resuspension in PBS, cells were imaged using a confocal microscope (Nikon A1R).
For flow cytometry analysis, human IgG or D‐PCF‐treated cells were washed twice with PBS and stained with eFluor870 fixable viability dye (ThermoFisher Scientific) for 20 min at 4 °C. The cells were then fixed with Fix/Perm buffer for 20 min at 4 °C, permeabilized with 0.1% Triton X‐100 for 10 min and stained with anti‐human IgG‐FITC for 1 h at 4 °C. Flow cytometry acquisition was performed using the Cytoflex (Beckman Coulter) and analysed using FlowJo V10.
9
Characterizing Conjunctival Immune Cells
10
Multicolor FACS Analysis of Tumor-Specific T Cells
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