Fluorescent dye-labeled antibodies specific for CD4, CD25, CD11b, Gr-1 (clone RB6-8C5), Foxp3, GATA3, B220, NK1.1, CD19, CD3, IL-5, and IL-13 were purchased from eBioscience and Biolegend. Fc block (2.4G2) was purchased from BD. Dead cells were stained by fixable aqua dead cell staining kit. Intracellular staining for Foxp3 and GATA3 was performed by Intracellular Fixation and Permeabilization Buffer Set (eBioscience). Intracellular staining of IL-5 was performed after restimulation with PMA, ionomycin and brefeldin A for 5 h. Restimulated cells were treated with Intracellular Fixation and Permeabilization Buffer Set (eBioscience) and stained with IL-5 antibodies. Flow cytometric analysis was performed on a Beckman Coulter Cytoflex flow cytometer and analyzed by Flowjo software (Tree Star, Ashland, OR).
Intracellular fixation and permeabilization buffer set
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Intracellular Fixation and Permeabilization Buffer Set is a laboratory equipment designed for the fixation and permeabilization of cells prior to intracellular staining and flow cytometry analysis. The set includes a fixation buffer and a permeabilization buffer to prepare cell samples for the detection of intracellular proteins and other targets.
Automatically generated - may contain errors
Lab products found in correlation
Cell stimulation cocktail, by Thermo Fisher Scientific (13 mentions)
Facscanto 2, by BD (11 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (8 mentions)
Brefeldin a, by Thermo Fisher Scientific (7 mentions)
Facscalibur flow cytometer, by BD (6 mentions)
Ionomycin, by Merck Group (5 mentions)
Fcr blocking reagent, by Miltenyi Biotec (5 mentions)
Lsrfortessa x 20, by BD (5 mentions)
Cytoflex s, by Beckman Coulter (4 mentions)
Lsr 2, by BD (4 mentions)
Facsdiva, by BD (4 mentions)
Anti foxp3, by Thermo Fisher Scientific (4 mentions)
Lsrfortessa, by BD (4 mentions)
Facsaria 2, by BD (4 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (3 mentions)
Protein transport inhibitor, by Thermo Fisher Scientific (3 mentions)
Collagenase 4, by Worthington (3 mentions)
Anti il 17a, by Thermo Fisher Scientific (3 mentions)
Anti cd4, by Thermo Fisher Scientific (3 mentions)
Protein transport inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
116 protocols using intracellular fixation and permeabilization buffer set
1
Multiparameter Flow Cytometric Analysis
2
Splenic T Cell Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens were harvested on the day of euthanasia to prepare single cell suspensions via passage through mesh screens. The splenocytes were washed with red blood cell lysis buffer (BioLegend, USA) and then washed twice with PBS. To perform intracellular labelling, cells (2 × 106 per sample) were incubated with Cell Stimulation Cocktail (eBioscience, USA) for 5 h at 37 °C. Subsequently, the cells were surface-stained with a FITC-labelled CD4 monoclonal antibody (eBioscience), then fixed and permeabilized using an Intracellular Fixation and Permeabilization Buffer Set (eBioscience), followed by intracellular staining with a PerCPCy5.5-labelled IFN-γ monoclonal antibody (eBioscience) and an APC-labelled IL-17A monoclonal antibody (eBioscience). To determine Treg frequency, the cells (2 × 106 per sample) were surface-stained with a FITC-labelled CD4 monoclonal antibody (eBioscience) and an APC-labelled CD25 monoclonal antibody (eBioscience). After fixation with the Intracellular Fixation and Permeabilization Buffer Set (eBioscience), the cells were stained with a PE-labelled FoxP3 monoclonal antibody (eBioscience). Isotype-matched IgG antibody (eBioscience) was used as a negative control. The stained cells were analysed using a FACSCalibur flow cytometer (BD Biosciences, USA) with FlowJo software version 7.6.
3
Osteopontin and CCL13 Expression in iTF-Microglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
iTF-Microglia were treated for 6 h with 1:2,000 GolgiPlug (BD; Cat. No. 555029) or dimethylsulfoxide as control before dissociating. Cells were fixed and permeabilized with the eBioscience Intracellular Fixation and Permeabilization Buffer Set (Invitrogen; Cat. No. 88-8824-00) according to the manufacturer’s instructions. Cells were stained with 1:75 Anti-Hu Osteopontin (SPP1) eFluor 660 (eBioscience; Cat. No. 50-9096-42) or 1:75 Human CCL13 488 (R&D Systems; Cat. No. IC327G) or their isotype controls Mouse IgG1 Control Alexa Fluor 488 conjugated (R&D Systems; Cat. No. IC002G) and Mouse IGG1 kappa Isotype (eBioscience; Cat. No. 50-4714-82) overnight at 4 °C. Cells were washed twice with DPBS before analyzing them by flow cytometry using the BD FACS Celesta (BD Biosciences) or the BD FACSAria Fusion using BD FACSDiva (v.8.0.1.1) software. Flow cytometry data were analyzed using FlowJo, raw median fluorescence intensity values of Osteopontin (SPP1) and CCL13 stained cells were normalized to isotype-control samples and data were plotted as fold change using Prism 8. The gating strategy used to determine the percentage of SPP1-positive cells is shown in Supplementary Fig. 1c .
4
Multiparameter Analysis of Lymphocyte Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly isolated lymphocytes from the peripheral blood were stimulated with PMA/ionomycin (Biolegend) combined with Brefeldin A (Invitrogen) for 4 h. The cells were collected after stimulation and stained with the following antibodies: APC-anti-CD16 + 56, PerCP-Cy5.5-anti-CD3, and Alexa Fluor 750 anti-CD8 (Toimmy Biotech). Using the Intracellular Fixation and Permeabilization Buffer Set (Invitrogen), the cells were fixed, permeabilized, and stained for intracellular cytokine staining with PE-Cy7-anti-IFNγ and FITC-anti granzyme B (Toimmy Biotech). The cells were then analyzed with DxFLEX flow cytometry.
In addition, the levels of cytokines IL-2, IL-4, IL-6, IL-10, IFN-γ, and IL-17A in the plasma were determined by cytometric bead array (BD Biosciences), and the plasma IL-12 levels were measured by ELISA as per the manufacturer's directions.
In addition, the levels of cytokines IL-2, IL-4, IL-6, IL-10, IFN-γ, and IL-17A in the plasma were determined by cytometric bead array (BD Biosciences), and the plasma IL-12 levels were measured by ELISA as per the manufacturer's directions.
5
Multiparametric Immune Profiling of Single-Cell Tumor Suspensions
6
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Surface and intracellular staining for flow cytometry analysis were performed as described previously (23 (link), 26 (link)). The antibodies used for surface staining included APC-Cy7-anti-CD4, Pacific Blue-anti-CD8a, APC-anti-CD44, APC-anti -PD-1, PerCP-Cy5.5-anti-CD43, PE-Cy7-anti-CD62L, PE-anti-CTLA-4, PE-anti-LAG-3 (all from BD Biosciences), PE-anti-CD45.1, PE-anti-CD45.2, and PE-Cy7-anti-CD45.2 (all from BioLegend). For intracellular cytokine staining, cells were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (Invitrogen) and were stained with APC-anti-interferon (IFN)γ, PerCP-Cy5.5-anti-interleukin (IL)-2, FITC-anti-tumor necrosis factor (TNFα) (from BD Bioscience). For Ki67 staining, cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (Biolegend) and were stained with BV421-anti-Ki-67 (Biolegend). Freshly isolated cells were used for all assays and about 20,000–40,000 T cells were acquired for each sample using BD FACSCanto II flow cytometer. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506 (eBioscience).
7
Isolation and Characterization of Liver Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lymphocytes in the liver were isolated as described in a previous study (Ma et al., 2017 (link)). Briefly, the mouse liver was perfused with PBS and then digested with an enzyme solution containing 0.05% collagenase type IV (Sigma-Aldrich), 0.002% DNAase I (Sigma-Aldrich), and 10% fetal bovine serum for 30 min. Lymphocytes in the homogenate were isolated using Percoll (Sigma-Aldrich), following the manufacturer's instructions and cultured in RPMI 1640 medium in 96-well plates and stimulated with CD8+ T cell epitope (Kb-HBV Cor93−100 epitope, MGLKFRQL, 10 μg/mL). For cell surface staining, the cells were stained with BV421-anti-CD8 (eBioscience, San Diego, CA, USA). For intracellular cytokine staining, the cells were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (Invitrogen, Carlsbad, CA), and then stained with the following antibodies: APC-anti-IFN-γ, PE-anti-IL-2, and FITC-anti-TNF-α (Biolegend, San Diego, CA, USA). All the samples were stained with Fixable Viability Dye eFluor 506 (eBioscience) to exclude dead cells. The stained cells were analyzed using a BD FACSCanto II flow cytometer. Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
8
Lung and Snout Tissue Digestion Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lungs and snouts were cut into small pieces and digested in RPMI containing 1 mg/mL collagenase IV (cat. number LS004189, Worthington Biochemical Corporation), 40 mg/mL DNase I, and 2% FCS for 30 min at 37°C. Any tissue remaining after 30 min was further digested with 1 mg/mL collagenase D (cat. number 11088882001, Roche), and 40 mg/mL DNase I and 1% of FCS for 20 min at 37°C. The reaction was stopped by addition of 5 mmol/L EDTA and 10% BSA. Samples were further disaggregated through a 70-mm cell strainer and blocked with anti-CD16/32 (cat. number 14-0161-86, Invitrogen). Single-cell suspensions were counted and stained with antibodies as previously described (71 (link)). Intracellular staining with anti-CD68 was done using the Intracellular Fixation and Permeabilization buffer set (Thermo Fisher): Fixation/permeabilization concentrate (cat. number 00-5123-43) and Diluent (cat. number 00-5223-56), Permeabilization Buffer 10 × (cat. number 00-8333-56). Cells were acquired on a Fortessa and analyzed using FlowJo software using the gating strategy depicted in Fig. S2A.
9
Flow Cytometry Analysis of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry was performed on a LSR II system (BD Biosciences) and data were analyzed using FlowJo software (Tree Star, Inc, Ashland, OR). For cell surface staining: cells were stained with anti-mouse purified CD16/32 (clone 93), BV 510-CD45 (clone 30-F11), BV 421-TCRβ (clone H57-597), AF 700-CD3 (clone 17A2), PerCP/Cy5.5-CD4 (clone RM4-5) and BV 711-CD8 (clone 53–6.7) (BD Biosciences). The live cells were discriminated by Zombie NIR Fixable Viability Kit (Biolegend). For intracellular staining, cells were incubated with cell activation cocktail (Biolegend) in the presence of Monensin Solution at 37°C for 12 hr. Anti-mouse PE-IL-17A (clone eBio17B7) was added after cell fixation and permeabilization with Intracellular Fixation and Permeabilization Buffer Set (Thermo Fisher).
10
Characterization of Murine Bone Marrow-Derived Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDMs were harvested from the 6 well plate using Versene solution and immediately washed with PBS containing 2% FBS prior to immunostaining. Cells suspensions were stained with TruStain FcX (101319, Biolegend) and fixable viability dye eFluor 780 (65-0865-14; Thermo Fisher Scientific, MA, USA). The following surface marker antibodies and appropriate isotypes, all purchased from Biolegend unless otherwise specified, were used: Pacific Blue-anti-CD11b (M1/70), FITC-anti-F4/80 (BM8, Invitrogen), Per-CP-Cy5-5anti- I-A/I-E (M5/114.15.2), PE-Cy7-anti-CD115 (AFS98), and APC-anti-CD206 (FVS660, eBioscience). Cells were fixed and permeabilized using eBioscience intracellular fixation and permeabilization buffer set (88-8824-00; Thermo Fisher Scientific, MA, USA) following manufacturer's instructions. Following permeabilization, cells were stained with anti-CD206 (FVS660, eBioscience) for intracellular antigen detection. Cells were washed with PBS containing 2% FBS. Data was acquired using CytoFLEX S (Beckman Coulter, USA) and analyzed using Flowjo software.
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!