Viability dye and antibodies against mouse CD4, CD8, CD90.2, CTLA4, CD39, CD73 (biolegend), CD25, CD44, CD62L, Foxp3, T-Bet, Gata-3, Helios, IFN-γ, IL-4, IL-17A, IL-2, IL-10, OX40, Nrp1 (eBioscience), p65, pS536 p65 (Cell signaling Technology) were used. Cell suspensions were stained for surface markers and viability dye for 30 min on PBS/0.5%FCS. Foxp3, Helios, and CTLA4 staining was performed overnight using the ebioscience Cytofix/CytopermTM kit. For cytokine detection, cell suspensions were pre-incubated with 50 ng/mL PMA, 500 ng/mL ionomycin and 10 µg/mL brefeldin A for 4h in complete medium. Following CD16/32 blocking with specific mAbs, the cells were surface stained for the indicated markers then permeabilized and stained intracellularly overnight with mAbs against IL-4, IL-10, IFN- γ or IL-17 using the ebioscience Cytofix/CytopermTM kit. For pS536p65, and p65 staining, spleen cells were fixed with PBS/2% paraformaldehyde for 20min, permeabilized in 90% methanol for 30 min on ice and stained for CD4, pS536p65 and p65 in PBS. All flow cytometry acquisitions were performed on a BD Fortessa cytometer using DIVA software (BD Biosystems) and analyzed using FlowJo Version 10 (Tree Star). All mouse antibodies used are listed in Supplementary Table 1 . The gating strategies for discriminating human and mouse Treg and Teff cells are shown in Fig. S9 .
Cytofix cytoperm kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Cytofix/Cytoperm kit is a laboratory product designed for the fixation and permeabilization of cells. It provides a solution for preparing samples for intracellular staining and flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (7 mentions)
Brefeldin a, by Merck Group (5 mentions)
Phorbol myristate acetate (pma), by Merck Group (5 mentions)
Anti cd4 fitc, by Thermo Fisher Scientific (3 mentions)
Anti cd25 apc, by Thermo Fisher Scientific (3 mentions)
Anti foxp3 pe cy7, by Thermo Fisher Scientific (3 mentions)
Anti il 4 pe, by Thermo Fisher Scientific (3 mentions)
Canto software, by BD (3 mentions)
Anti ifn γ pe cy7, by Thermo Fisher Scientific (3 mentions)
Facscanto 2 cytometer, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Anti il 10 pe, by Thermo Fisher Scientific (2 mentions)
Monensin, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Apc conjugated anti il2 jess 5h4, by Thermo Fisher Scientific (2 mentions)
Pe cy7 conjugated anti ifnγ xmg1.2, by Thermo Fisher Scientific (2 mentions)
38 protocols using cytofix cytoperm kit
1
Comprehensive Immune Cell Analysis Protocol
2
Multiparameter Analysis of iNKT and NK Cells
3
T-Cell Cytokine Profiling by ELISA and Flow Cytometry
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After 72 h of polarization, cell culture supernatants were assayed by ELISA to measure the levels of mouse IFN-γ, IL-4, IL-9, and IL-17A (BioLegend) according to the manufacturer’s instructions. For intracellular staining, cells were cultured for 3 days, restimulated for 1 additional day, and then stimulated for 4 h at 37 °C in RPMI-1640 medium containing phorbol 12-myristate 13-acetate (50 ng ml−1, Sigma-Aldrich, St Louis, MO, USA) and ionomycin (1 μg ml−1, Sigma-Aldrich). After staining for surface markers, the cells were fixed and permeabilized according to the manufacturer’s instructions (Cytofix/Cytoperm Kit, Thermo Fisher Scientific) and then stained for intracellular products. The following monoclonal antibodies were used for the flow cytometric analyses: fixable viability dye eFluorTM 450- or phycoerythrin-conjugated anti-CD4 and allophycocyanin-conjugated anti-IFN-γ, anti-IL-4, anti-IL-9, anti-IL-17A, or anti-Foxp3. All events were acquired on a BeckmanCoulter DxFLEX flow cytometer equipped with CytExpert experiment-based software (BeckmanCoulter, Inc.), and data were analyzed using FlowJo software (TreeStar). Gating strategies were presented in Supplementary Fig. 9 .
4
Quantification of RBM17 Expression in AML
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Primary AML cells and OCI-AML-8227 cells were initially stained with anti-CD34 APC (555824, BD Biosciences) antibody and LIVE/DEAD Fixable Green (L34969, Thermo Fisher) and then fixed with the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Fixed and permeabilized cells were immunostained with anti-RBM17 rabbit antibody (ab204333, Abcam) and detected by Alexa-Fluor 405 goat anti-rabbit IgG(H + L) secondary antibody (Thermo Fisher) through FCAS. For RBM17 knockdown efficiency test, sorted cells were initially stained with LIVE/DEAD Fixable Near-IR (L34975, Thermo Fisher) for 30 minutes and then fixed with the Cytofix/Cytoperm kit. Fixed and permeabilized cells were immunostained with anti-RBM17 rabbit antibody and detected the same way as described above.
5
Isolation and Characterization of Murine Macrophages
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Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, LPS, and trypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gentamicin, Fungizone®, and IL-4 were bought from Thermo Fisher Scientific (Waltham, MA, USA). IFN-γ, TNF-α, IL-1β, IL-6, IL-10, and TGF-β were bought from R&D Systems (Minneapolis, MN, USA). Antibodies used for flow cytometry: CD45-APCeFluor780, CD11b-AF700, MHC-I-APC, PD-L1-PE-Cy7, Arg-1-eFluor450, and iNOS-PE were procured from Thermo Fisher Scientific while MHC-II-BV510 was purchased from Biolegend (San Diego, CA, USA). Cytofix/Cytoperm kit used for cell permeabilization was also purchased from Thermo Fisher Scientific.
6
Flow Cytometry Immunostaining Protocol
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The BD-Pharmigen Fc block (2.5 µg/106 cells) was used to minimize the nonspecific binding of immunoglobulins to Fc receptors before immunostaining (18 (link)). PBMCs were resuspended at a concentration of 2 × 106/mL. A total of 5–10 µL (in accordance with manufacturer's instructions) of mouse mAb (Supplementary Materials and Methods ) were added to 50 µL of PBMC suspension, as described previously (18 (link)), and incubated for 15 minutes in the dark at room temperature (20°C–25°C). Next, 200 µL of a fixation/permeabilization buffer (BD-Pharmingen Cytofix/Cytoperm Kit) for intracytoplasmic staining and Transcription Factor Staining Buffer Set (eBioscience, Thermo Fisher Scientific) for nuclear staining were added to each tube and incubated for 20 minutes in the dark at 4°C. The antibodies used for intracellular staining are listed in Supplementary Materials and Methods . For each staining, an Ig isotype-conjugated antibody was used as a control for nonspecific binding. Samples were acquired using a BD Accuri C6 Cytometer (Becton, Dickinson and Company BD) and analyzed using FlowJo or C6 Accurì software.
7
Cytokine Profiling in Candida Infection
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Mice were injected intravenously with 2 × 105C. albicans train SC5314 yeast. After 5 days, splenocytes were obtained and stimulated with heat-killed C. albicans (MOI, 1) for 48 h. For cytokine detection, supernatants were collected to measure the concentrations of IFN-γ and IL-17A by ELISA. For intracellular staining, cells were stimulated with Cell Activation Cocktail (with Brefeldin A) (Biolegend) for the final 6 h of incubation and then were treated with a Cytofix/Cytoperm kit (eBioscience). The expression of intracellular factors was analyzed by flow cytometry.
8
Evaluating T-cell Subsets in Asthmatic Mice
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To evaluate the recruitment of Th1, Th2, and Tregs induced by ASCs treatment, LLN cells from OVA-induced asthmatic mice and ASCs-treated asthmatic mice were cultured in anti-CD3-coated plate for 6 h. To evaluate To evaluate CD4+CD25+Foxp3+ (Tregs) and IL-10+/CD4+ T cells, cells were stained with anti-CD4-FITC (0.5 mg/ml) and anti-CD25-APC (0.2 mg/ml) in accordance with the manufacturer’s recommendations (eBiosciences, San Diego, CA). After surface staining, the cells were permeabilized using a Cytofix/Cytoperm Kit (eBiosciences). After permeabilization, the cells were stained with anti-Foxp3-PE-cy7 or anti-IL-10-PE (eBiosciences).
To assess the Th1 and Th2 cell population, LLNs cells were stained with an anti-CD4-FITC Ab. After surface staining, the CD4+ T cells were stained with intracellular anti-IFN-γ-PE-cy7 (eBiosciences) and anti-IL-4-PE (eBiosciences) Abs. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
To assess the Th1 and Th2 cell population, LLNs cells were stained with an anti-CD4-FITC Ab. After surface staining, the CD4+ T cells were stained with intracellular anti-IFN-γ-PE-cy7 (eBiosciences) and anti-IL-4-PE (eBiosciences) Abs. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
9
Analyzing T Cell Subsets in Asthma Model
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To evaluate the recruitment of Th1, Th2, and Treg induced by ASC sup treatment, the LLN cells of the OVA-induced animal model of acute asthma and ASC sup-treated animal model of acute asthma were cultured on anti-CD3-coated plates for 6 hours. To determine the CD4+CD25+Foxp3+ (Treg) and IL-10+/CD4+ T cell populations, the cells were stained with anti-CD4-FITC (0.5 mg/ml) and/or anti-CD25-APC (0.2 mg/ml) in accordance with the manufacturer’s recommendations (eBioscience, San Diego, CA, USA). After surface staining, the cells were permeabilized using a Cytofix/Cytoperm kit (eBioscience). After permeabilization, the cells were stained with anti-Foxp3-PE-cy7 or anti-IL-10-PE (eBioscience). To quantify the Th1 and Th2 cell populations, the LLN cells were stained with an anti-CD4-FITC antibody. After surface staining, the CD4+ T cells were stained with intracellular anti-IFN-γ-PE-cy7 (eBioscience) and anti-IL-4-PE (eBioscience) antibodies. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
10
Multiparameter Analysis of Murine Immune Cells
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