PBMCs, either isolated from the peripheral blood or harvested after a 5-day MLC, were analyzed via cell-surface staining using CD3 (SP34), CD4 (L200), CD8 (SK1), CD20 (2H7), CD25 (M-A251) (all BD Pharmingen), CD16 (NKP15, BD Biosciences), and NKG2a (Z199, Beckman Coulter) antibodies. For chimerism analyses, we used an anti–MHC class I HLA mAb (H38, One Lambda, Inc.) that reacts specifically with an MHC class I antigen on donor but not recipient cells. To assess intracellular protein expression of FOXP3, cells were permeabilized using Fixation/Permeabilization solution (eBioscience) and then stained with anti-FOXP3 mAb (PCH101, BD Pharmingen). Cells were analyzed on a FACSverse (BD Biosciences) or Accuri Flow Cytometer (BD Biosciences) using FlowJo software (FLOWJO LLC).
Fixation permeabilization solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fixation/permeabilization solution is a laboratory reagent used to prepare cells for analysis by flow cytometry or microscopy. It serves to fix the cellular structure and permeabilize the cell membrane, allowing access to intracellular antigens or other targets for staining and detection.
Automatically generated - may contain errors
Lab products found in correlation
Permeabilization buffer, by Thermo Fisher Scientific (13 mentions)
Brefeldin a, by Thermo Fisher Scientific (8 mentions)
Ionomycin, by Merck Group (8 mentions)
Lsrfortessa, by BD (6 mentions)
Facsaria cell sorter, by BD (5 mentions)
Golgiplug, by BD (4 mentions)
Diva software, by BD (4 mentions)
Facscalibur flow cytometer, by BD (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (4 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Ifn γ, by BioLegend (3 mentions)
Brefeldin a, by Merck Group (3 mentions)
Facscan, by BD (3 mentions)
Golgistop, by BD (3 mentions)
Facsverse, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Cd57 clone nk 1, by BD (2 mentions)
Klrg 1 clone 13f12f2, by Thermo Fisher Scientific (2 mentions)
Tim 3 clone 344823, by R&D Systems (2 mentions)
67 protocols using fixation permeabilization solution
1
Multiparametric Flow Cytometry Analysis
2
Flow Cytometric Analysis of PGC-1β in Monocytes
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Peripheral blood CD14+ monocytes were stained first with PE‐conjugated anti‐CD14 monoclonal antibody (BD Biosciences) for cell surface antigen. Cells were washed twice with staining wash buffer and centrifuged (1,000 revolutions per minute for 5 minutes) to pellet the cells. They were then resuspended with 100 μl of fixation/permeabilization solution (eBioscience) for 30 minutes at 4°C to expose intracellular antigen. Cells were washed twice with 500 μl of wash buffer and suspended with 100 μl of permeabilization buffer mixed with 1 μl of rabbit anti‐human/mouse PGC‐1β antibody (Bioss) in the dark for 30 minutes at room temperature. Next, they were washed twice and resuspended with 100 μl of permeabilization buffer mixed with 1 μl of FITC‐conjugated anti‐rabbit secondary antibody (Invitrogen) in the dark for 20 minutes at room temperature. Stained cells were washed with permeabilization buffer and resuspended with 200 μl of phosphate buffered albumin (0.5% BSA and 0.05% NaN3 in PBS) before flow cytometric analysis. In each case, staining was compared to that of the appropriately labeled isotype control antibody.
3
Teriflunomide Effects on HAM/TSP PBMCs
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PBMCs of patients with HAM/TSP were cultured at 3 × 105 cells/well in 96-well round bottom microplates with defined concentrations of teriflunomide (0, 25, 50, and 100 μM). After 20 hours, the cells were stained with antibodies for CD3, CD4, CD8, and CD25 (all from BD Biosciences). After treatment with fixation/permeabilization solution (eBiosciences), the cells were stained with antibody for Tax (Lt-4). The data were acquired on an LSRII flow cytometer (BD Biosciences) and were analyzed using FlowJo 10.5 software (FlowJo LLC).
4
Multiparametric Flow Cytometry Analysis
5
Multiparametric phenotyping of lymphocytes
6
Splenic Immune Cell Phenotyping
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Single-cell suspensions were prepared from fresh spleen tissue and followed by lysis of red blood cell (RBC) using ACK lysis buffer. For surface staining, 1 × 106 splencytes per 100 μl were incubated for 30 min at 4 °C with the following fluorescently labeled conjugated mAbs: CD4-FITC (eBioscience, San Diego, CA), CXCR5-PE (eBioscience), CXCR5-APC (eBioscience), ICOS-PE-Cy5.5 (eBioscience), OX40-APC (eBioscience), CD40L-APC (eBioscience), PD-1-APC (eBioscience), CD19-APC (eBioscience), IL-21R-PE (BioLegend). After staining of surface markers, the cells were permeabilized with fixation/permeabilization solution (eBioscience) and subsequently intracellularly stained with Bcl-6-PE (BD Pharmingrn, San Jose, CA), the cells were then wash twice using permeabilization buffer, and eventually fixed with 4% paraformaldehyde in PBS. For APC-conjugated anti-IL-21(BD Pharmingrn), spleen cells were stimulated with PMA and Ionomycin for five hours and subsequently intracellularly stained, all procedures were in accordance with Bcl-6 staining method. In addition, Tfh (CD4+CXCR5+) subsets and B (CD19+) subsets were sorted with a FACS Aria cell sorter (BD Bioscience).
7
Immunophenotyping of Activated PBMCs
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Freshly thawed or stimulated PBMCs were centrifuged at 300g for 5 minutes, resuspended in phosphate-buffered saline (Sigma-Aldrich, St. Louis, MO) supplemented with 2% heat-inactivated fetal bovine serum (GE Healthcare/PAA, Little Chalfont, UK) and 2 mM EDTA (Sigma-Aldrich) with fluorochrome-conjugated antibodies at the indicated working concentrations (see table e-1 at Neurology.org/nn ) or isotype-matched controls, and incubated at 4°C for 30 minutes. Staining of chemokine receptors was performed at 37°C for 30 minutes. Subsequently, cells were washed twice and either analyzed by flow cytometry (Navios; Beckman Coulter, Brea, CA) or stained for intracellular proteins with fixation/permeabilization solution (eBioscience, San Diego, CA) following the manufacturer's instructions. Resulting data were analyzed using Kaluza Flow Cytometry Analysis software version 1.2 (Beckman Coulter) and Prism software version 5.04 (GraphPad, La Jolla, CA).
8
Isolation and Characterization of Mouse Colonic Immune Cells
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Mouse lamina propria mononuclear cells (LPMCs) were obtained from the colon as described previously 7 (link). Cells were treated with a cocktail containing phorbol 12-myristate 13-acetate (PMA), ionomycin, Brefeldin A, etc. (eBioscience, 004975) for 5 h in the cell culture incubator at 37 °C. Subsequently, Fc block solution was added to block non-specific Fc-mediated interactions. Cells were stained for surface markers with monoclonal antibodies against CD4 (11-0041-82), CD25 (12-0251-81), and then fixed and permeabilized with Fixation/Permeabilization solution (eBioscience, 005523). Finally, intracellular/intranuclear cytokines were stained with monoclonal antibodies against IL-10 (12-7101-82), IL-17A (17-4321-81) and Foxp3 (17-5773-80), followed by flow cytometry analysis (BD FACSVerseTM, USA). All the antibodies were purchased from eBioscience.
9
Immune Cell Extraction and Analysis
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Mononuclear cells were extracted by mashing tumors or organs through a cell strainer with a syringe plunger. BM cells were flushed out of decapped long bones using a syringe. Single-cell suspensions (1–2 × 106 for spleen, 5 × 106 for BM) or whole blood (50 μl) were stained with antibody combinations as per Supplementary Table S1 . To assess proliferation, 3 × 106 splenocytes purified and stained for GC markers were treated with fixation/permeabilization solution (eBioscience, cat. #00-5523) for 1 h at 4°C in the dark, followed by 1 h incubation with anti-Ki67-PECY7 (eBioscience) at 4°C. To assess apoptosis, 106 splenocytes were incubated with FITC-conjugated CaspGLOW pan-caspase substrate (BioVision) for 60 min at 37°C in warm culture media followed by surface marker staining and flow cytometry analysis. Data were acquired using FACS Caliber 2, BD LSR I or BD LSR Fortessa (BD Biosciences) and analyzed using FlowJo.
10
Quantifying Granzyme B in T Cells
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First, cells (around 105 cells) were stained with the cell surface markers CD103 (αE)-FITC, Clone Ber-ACT8, BD Pharmingen) and CD8-PerCP (clone SK1, BD Biosciences). Then, cells were fixed in a fixation/permeabilization solution (eBioscience) for 10 minutes. After washing in a permeabilization solution (eBioscience), cells were incubated with mAb anti-granzyme B-PE (2 µg/ml, clone CLB-GB11, Sanquin, Amsterdam, The Netherlands) in the permeabilization solution (eBioscience) for 30 minutes. Cells were washed with PBS2+ and FACS analysis was performed.
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