Decidual tissues were washed with saline, collected in ice-cold MACS Tissue storage solution (catalog no. 130-100-008, Miltenyi, Germany) and dissociated into single cells. CyTOF analyses were performed by PLT Tech Inc. (Hangzhou, China). In brief, cells were stained 5 min with 5 µM 194Pt cisplatin (Fluidigm) for viability in phosphate buffered saline (PBS). After being blocked with PBS containing 5% goat serum and 30% bovine serum albumin (BSA) for 30 min at 4°C, cells were stained with cell-surface antibodies for 30 min at 4°C and washed twice with PBS containing 2.5% BSA. The Foxp3 Fixation and Permeabilization kit (eBioscience) was used according to the instructions of the manufacturer, and cells were incubated overnight in 1.6% paraformaldehyde (PFA) PBS with 100 nM iridium nucleic acid intercalator (Fluidigm) and lastly incubated with intracellular antibodies in permeabilization buffer for 30 min at 4 C after being washed twice with Foxp3 permeabilization buffer and twice with FACS buffer. Cells were washed, resuspended at a concentration of one million cells/ml with water containing EQ Four Element Calibration Beads (Fluidigm), and analyzed within 12 h on a Helios CyTOF System (Fluidigm) at an event rate of <500 events/s.
Foxp3 fixation and permeabilization kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FoxP3 fixation and permeabilization kit is a laboratory tool designed for the preparation of cells for intracellular staining and flow cytometric analysis of the FoxP3 transcription factor. The kit provides a gentle and effective method for the fixation and permeabilization of cells, allowing for the detection of intracellular proteins such as FoxP3.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii flow cytometer, by BD (5 mentions)
Efluor 506, by Thermo Fisher Scientific (4 mentions)
Lsr 2, by BD (3 mentions)
Anti cd43 1b11, by BioLegend (3 mentions)
Brefeldin a, by BD (2 mentions)
Efluor 780, by Thermo Fisher Scientific (2 mentions)
Anti cd19 1d3, by BioLegend (2 mentions)
Anti cd86 gl 1, by BioLegend (2 mentions)
Ki67 sola15, by BD (2 mentions)
Iridium nucleic acid intercalator, by Standard BioTools (1 mentions)
194pt cisplatin, by Standard BioTools (1 mentions)
Helios cytof system, by Standard BioTools (1 mentions)
Macs tissue storage solution, by Miltenyi Biotec (1 mentions)
Eq four element calibration beads, by Standard BioTools (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Guava easycyte plus, by Merck Group (1 mentions)
Anti cd4 pe clone gk1.5, by Thermo Fisher Scientific (1 mentions)
28 protocols using foxp3 fixation and permeabilization kit
1
Decidual Tissue Single-Cell CyTOF Analysis
2
Intracellular Staining for Bcl6 and Foxp3
3
Flow Cytometry Analysis of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the antibodies used for flow cytometry were purchased from eBioscience, Biolegend and BD biosciences. For cell surface staining, a lineage cocktail consisting anti-mouse CD3 (clone 145-2C11), anti-mouse Ly-6G/Ly-6C (clone RB6-8C5), anti-mouse CD11b (clone M1/70), anti-mouse CD45R/B220 (clone RA3-6B2), and anti-mouse TER-119/Erythroid cells (clone Ter-119) was used. FITC-streptavidin was used to stain biotin labelled primary antibody cocktail. Other antibodies used were rat anti-mouse CD45, anti-mouse CD127 (clone A7R34), anti-mouse CD90.2 (clone 53-2.1), anti-mouse KLRG1 (clone 2F1), and anti-mouse GATA3 (clone TWAJ). Foxp3 fixation and permeabilization kit (eBioscience) was used for intracellular staining.
4
Immunophenotyping of Basal Cell Carcinoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly excised 3 mm punch biopsies from 3 BCC lesions were collected in DMEM media. Tissue were transported to cell culture lab on ice and stored in DMEM media for 24–48 h at 4 °C. Cell suspensions were generated according to the following protocol87 (link). Cells were processed for surface labeling with anti-CD3, anti-CD45, anti-CD4, and anti-CD8 antibodies. Live cells are distinguished from dead cells by using the fixable dye eFluor506 (eBioscience, Thermo Fisher Scientific, MA, USA). They were further permeabilized using a FOXP3 fixation and permeabilization kit (eBioscience, Thermo Fisher Scientific, MA, USA) and stained for Ki-67, FOXP3, and granzymeB. Data were acquired using the Aurora Five Laser flow cytometer (Cytek Biosciences, CA, US). Data were analyzed with FlowJo software version 10.5.3. (Tree Star Inc. OR, USA)88 .
5
Multiparametric Analysis of Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells isolated from tumors were processed for surface labeling with appropriate antibodies. Fixable viability dye eFluor506 (eBioscience) was used to separate dead and live cells. Cells were further permeabilized using FoxP3 fixation and permeabilization kit (eBioscience) and stained for Ki-67, FoxP3, Granzyme B, and CD3. Data were acquired using the LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar).
6
Immunophenotyping of T Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T cell activation beads were removed and cells were washed with PBS containing 1% FBS and 2 mM EDTA. For evaluation of isolated CD4+ and CD8+ T cells, cells were blocked using normal rat serum for 15 min at room temperature and stained with anti-CD3-FITC (clone OKT3; eBioscience) and anti-CD4-PE (clone GK1.5; eBioscience) or anti-CD8-PE/Cy5 (clone 53–6.7; Biolegend) for 1 h to assess purity. Cells were fixed and permeabilized using the FoxP3 fixation and permeabilization kit from eBioscience according to the manufacturer's instructions. Subsequently, cells were incubated with the following anti-mouse antibodies in different combinations: FoxP3 (clone FJK-16s), Tbet (clone 4B10), GATA3 (clone TWAJ), RORγ (clone AFKJS-9), or isotype controls (all from eBioscience) for 1 h at room temperature. After washing, cells were analyzed by flow cytometry on a Guava EasyCyte Plus (Millipore) and analyzed using gauvaSoft Incyte software.
7
Intracellular Cytokine Staining and Tetramer Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Staining of transcription factors was performed after permeabilization with the FoxP3 Fixation and Permeabilization Kit (eBioscience). For intracellular cytokine staining, cells were stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) in 96-well U-bottom plates in complete media for 4–5 h, and brefeldin A (BD Biosciences) was added for the last 3–4 h. Intracellular staining was performed using the BD ICS kit as per the manufacturer’s instructions with overnight incubation (4°C) of permeabilized cells with transcription factor–specific antibodies. Antibodies are listed in Table S3 . Recombinant HA probe for B cells was generated as previously described (Angeletti et al., 2017 (link)). The HA I-A(b) tetramer (PR8 HA 91–107 RSWSYIVETPNSENGIC; Miller et al., 2015 (link)) and the NP I-A(b) tetramer (NP 311–325 QVYSLIRPNENPAHK; Botta et al., 2017 (link)) were produced by the National Institutes of Health Tetramer Core Facility. Flow cytometry data were acquired on a BD LSRII with FACSDiva software and were analyzed with FlowJo software (Tree Star).
8
Multiparametric Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In general, cell surface staining was performed in ice-cold PBS containing 0.5% BSA for 30 min at 4 °C. Non-specific staining was blocked using Fc receptor blocker (BD Biosciences). Dead cells were excluded using the fixable viability dye eFluor780 (eBioscience, USA). Transcription factor staining was performed after using the Foxp3 Fixation and Permeabilization Kit (eBioscience). Surface molecule staining was performed before fixation. For intracellular cytokine staining, cells were stimulated in vitro for 6 h with Leukocyte Activation Cocktail (BD Biosciences) and stained according to the manufacturer's instructions. The following antibodies were used: live/dead (FVS780), anti-CD45 (HI30, 13/2.3), anti-CD4(RM4.5), anti-CXCR2 (SA0446), anti-CD3ε (152,303), anti-CD8α (53–6.7), anti-CD11c (B-ly6), anti-CD86 (CL-1), anti-G4S linker (GS-ARAP25), anti-Gr-1 (RB6-8C), anti-CD206 (C068C2), anti-CD62L (MEIL-14), anti-CD44 (IM-7)and isotype controls (MOPC-21,27–35,X40,R35-95). All antibodies were purchased from BD Bioscience and Biolegend (San Diego, USA).
9
Neutrophil HMGB1 Expression Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A single cell suspension was prepared from spleen and red blood cells were lysed. To identify neutrophils, cells were incubated with anti-CD11b (eBioscience, clone M1/70) to label monocytes and anti-CD11b plus anti-Ly6G (BD Biosciences, clone 1A8) to label granulocytes. Cells were washed, fixed and permeabilized with Foxp3 Fixation and Permeabilization kit, eBioscience) followed by incubation with anti-HMGB1 (Biolegend, clone3E8). Data was acquired with a Becton Dickinson LSRII instrument.
10
Comprehensive Tumor-Infiltrating Lymphocyte Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells isolated from tumours or tumour-draining lymph nodes were processed for surface labelling with several antibody panels staining CD45, CD3, CD4, CD8, CTLA-4, ICOS, CD11c, CD19, NK1.1, CD11b, PD-1, Ki-67, Tbet, RORγt, Bcl-6, GATA-3, EOMES and CXCR5. Fixable viability dye eFluor780 (eBioscience) was used to distinguish the live cells. Cells were further permeabilized using FoxP3 fixation and permeabilization kit (eBioscience), and stained for Ki-67, FoxP3, Granzyme B or IFNγ. Data were acquired using the LSRII Flow cytometer (BD Biosciences) and analysed using FlowJo software (Treestar). The quantifications of lymphocytes in all figures are based on CD45+CD3+CD11b− gate.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!