Intracellular cytokine capture for flow cytometric detection of IL-13 and IL-5 was carried out by restimulating samples with 50 ng of PMA (Sigma-Aldrich), 1 µM ionomycin (Sigma-Aldrich) in the presence of 1 µM monensin for 4 h in cDMEM at 37°C/5% CO2 . Unstimulated controls were cultured in cDMEM with monensin only. Following viability and surface marker staining, cells were fixed and permeabilized with the Foxp3 Staining Kit (eBiosciences). For analysis of exhaustion markers, including IL-10, samples were stimulated with 100 ng of PMA, 2 µM ionomycin for 3 h in the presence of 1 µM monensin, and 1× brefeldin-A (eBioscience) in cDMEM at 37°C/5% CO2. Unstimulated samples were cultured with monensin and brefeldin-A only. Fixation was carried out for 10 min with methanol-free 2% paraformaldehyde followed by permeabilization using the Foxp3 permeabilization buffer (eBiosciences). Pd-1 and Tigit were carried out as extracellular surface stains and Ctla4 was incorporated as both an extracellular and intracellular stain.
Foxp3 permeabilization buffer
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
FoxP3 permeabilization buffer is a laboratory reagent used to facilitate the intracellular staining of the transcription factor FoxP3. It is designed to permeabilize cell membranes, allowing for the detection and analysis of intracellular proteins by flow cytometry or other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 fixation buffer, by Thermo Fisher Scientific (5 mentions)
Lsrfortessa, by BD (5 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (3 mentions)
Ionomycin, by Merck Group (2 mentions)
Foxp3 pe cy7, by Thermo Fisher Scientific (2 mentions)
Facscanto flow cytometer, by BD (2 mentions)
Cytof, by Standard BioTools (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Corning (2 mentions)
Brilliant stain buffer, by BD (2 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (2 mentions)
Lsr fortessa 2, by BD (2 mentions)
Prism v8, by GraphPad (2 mentions)
Foxp3 fixation permeabilization buffer kit, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
21 protocols using foxp3 permeabilization buffer
1
Intracellular Cytokine and Exhaustion Marker Analysis
2
Intracellular Cytokine Profiling in Lymphoid Cells
3
Multicolor Flow Cytometry Analysis
4
Flow CyTOF Analysis of Endocrine Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow CyTOF was performed as described previously50 (link). Briefly, after isolating the dissociated cells, barcoding was conducted for donors following the manufacturer’s protocol (Fluidigm, 101-0804 B1). Following barcoding, metal-conjugated antibody labeling was carried out in ‘FoxP3 permeabilization buffer’ (eBioscience, 00-8333) with 1% FBS (Hyclone, Cat# 7207) for 12 hours at 4°C at a concentration of up to 3 million cells per 300 μl of antibody cocktail, followed by twice washing with FoxP3 permeabilization buffer. Cells were then incubated with the DNA intercalator Iridium (Fluidigm, 201192A) at a dilution of 1:4,000 in 2% paraformaldehyde (Electron Microscopy Sciences, 15714) in DPBS (Corning, 21-031-CV) at RT for 1 hr. Mass cytometry data were acquired by CyTOF (Fluidigm). Flow CyTOF data analyses of endocrine cell composition was performed using the Cytobank implement (https://www.cytobank.org/ ).
Normalized FCS files were pre-processed prior to TooManyCells analysis and visualization using FlowJo Version 10.6.1 by gating all events on singlets according to event length and DNA content and then on live cells based on cisplatin exclusion. The Singlet/Live gated population was exported to a CSV file for TooManyCells analysis. Two dimensional plots were visualized for combinations of individual channels.
Normalized FCS files were pre-processed prior to TooManyCells analysis and visualization using FlowJo Version 10.6.1 by gating all events on singlets according to event length and DNA content and then on live cells based on cisplatin exclusion. The Singlet/Live gated population was exported to a CSV file for TooManyCells analysis. Two dimensional plots were visualized for combinations of individual channels.
5
Flow Cytometry Immunophenotyping Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were thawed and counted. 1–2*10E6 cells were used for flow cytometry. Surface staining was performed in a total volume of 50 μl antibody master mix at RT for 15 min and washed twice before acquiring on BD LSR Fortessa. For intracellular staining, cells were permeabilized with FoxP3/Transcription Factor Staining Buffer Set (eBioscience) on ice for 30 min and washed twice with FoxP3 permeabilization buffer (eBioscience), followed by intracellular staining in a total volume of 50 μl antibody master mix on ice for 30 min. Cells were fixed with 2% PFA until measurement. Samples were then acquired and recorded on BD LSRFortessa™. For gating strategies see Additional file 1 : Fig. S5.
6
Neutrophil Isolation and Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Enriched human neutrophil fractions were isolated from heparinized blood from two healthy donors, as previously described (45 (link)). Briefly, after Ficoll-Paque separation (GE Healthcare), granulocytes were twice treated with red blood cell lysis using ACK lysing buffer (Thermo Fisher Scientific). Primary human neutrophils (0.2 × 106) were cultured in Hanks' balanced salt solution without calcium/magnesium for 4 h on 6 mm coverslips coated with 0.02% poly-L-Lysine (Sigma-Aldrich). Neutrophils were left untreated (medium) or stimulated with ionomycin (1 μM, Calbiochem) or phorbol 12-myristate 13-acetate (PMA) (50 nM, Calbiochem), then fixed with 3.7% formaldehyde and incubated in PBS-glycine (10 mM). Cells were permeabilized (eBioscience, Foxp3 permeabilization buffer), stained with murine chimeric ACPA IgG2a or control mAbs (10 μg/ml), and subsequently with anti-mouse-AlexaFluor488 (Invitrogen) and Hoechst (Thermo Fisher Scientific). Coverslips were mounted onto glass slides using Fluoromount-G (Southern Biotech). Images were acquired using a Leica TCS SP5 and a 63x oil objective. A z-dimension series was taken every 0.2 μm. Images were analyzed with the Fiji software.
7
Intracellular Cytokine Staining for Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The analysis was conducted according to the guidelines for the use of flow cytometry and cell sorting in immunological studies (Cossarizza et al., 2017 (link)).
For intracellular cytokine staining, cells were stimulated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml ionomycin (Sigma) for 1 hr followed by additional 3 hr in the presence of 5 µg/ml Brefeldin A (Sigma). Cell surfaces were stained with antibodies in the presence of 100 µg/ml 2.4G2 (anti-FcγRII/III; purified from hybridoma supernatants) to reduce unspecific antibody binding. Cells were fixed with 2% paraformaldehyde followed by an intracellular staining in 0.5% saponin. For intracellular staining of transcription factors, cells were fixed with FoxP3 fixation buffer (eBioscience) and stained in FoxP3 permeabilization buffer (eBioscience). Live cells were discriminated from dead cells with a fixable LIVE/DEAD stain (Life Technologies). The expression of phenotypic markers was determined with a BD LSR Fortessa (BD Biosciences) and analyzed with FlowJo (Treestar).
For intracellular cytokine staining, cells were stimulated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml ionomycin (Sigma) for 1 hr followed by additional 3 hr in the presence of 5 µg/ml Brefeldin A (Sigma). Cell surfaces were stained with antibodies in the presence of 100 µg/ml 2.4G2 (anti-FcγRII/III; purified from hybridoma supernatants) to reduce unspecific antibody binding. Cells were fixed with 2% paraformaldehyde followed by an intracellular staining in 0.5% saponin. For intracellular staining of transcription factors, cells were fixed with FoxP3 fixation buffer (eBioscience) and stained in FoxP3 permeabilization buffer (eBioscience). Live cells were discriminated from dead cells with a fixable LIVE/DEAD stain (Life Technologies). The expression of phenotypic markers was determined with a BD LSR Fortessa (BD Biosciences) and analyzed with FlowJo (Treestar).
8
Flow Cytometric Analysis of Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell suspensions were stained with fluorochrome labeled primary antibodies (see Supplementary Table 1 ) in Brilliant stain buffer (BD Biosciences) for 30 min on ice. Flow cytometry was performed on an LSR Fortessa II (BD Biosciences), FACSAria Fusion (BD Biosciences), or FACSMelody (BD Biosciences) and analyzed with FlowJo software (TreeStar). Dead cells were identified by staining with either 7-AAD (eBioscience) or Zombie UV fixable viability dye (BD Biosciences) and cell doublets were excluded on the basis of FSC-A/FSC-H. For intracellular staining, cells were stained for surface antigens, fixed with FoxP3 Staining Buffer set (eBioscience) and stained for αSMA in FoxP3 Permeabilization buffer (eBioscience). After washing, cells were stained with antibodies to surface antigens not compatible with fixation according to the manufacturer’s instructions.
9
Multicolor Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In cellular barcoding and Gata3 time course experiments, cells were harvested at indicated time points, washed once, and fixed in FoxP3 Fixation buffer (eBiosciences). Cells were permeabilized with FoxP3 permeabilization buffer (eBiosciences). Where applicable cells were barcoded by staining with Pacific Blue succinimidyl ester (Thermo Fisher / Molecular Probes), and washed. Cells were stained with antibodies to Gata3, Foxp3, and Bcl6 and analyzed by flow cytometry.
10
Intracellular Staining of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated cells were stained with antibodies for surface markers at room temperature for 15 min. Intracellular staining protocol was modified from eBioscience Foxp3 staining kit. Cells were fixed in 2% methanol free paraformaldehyde for 30 min at room temperature. Intracellular staining was done in Foxp3 permeabilization buffer (eBioscience) overnight at 4°. Cells were analyzed by flow cytometry on a BD Biosciences LSRFortessa. Data was analyzed using FlowJo 10 analysis software (BD Biosciences).
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!