Anti-IDH2 was from Abcam (# ab131263), anti-IDH1 was from Cell Signaling Technology (#8137S), and anti-HIF1α is from Novus biologicals (#NB100–105). Anti-mouse CD3 (clone 145–2C11, cat#16–0031), anti-mouse CD28 (clone 37.51, cat#16–0281), anti-mouse IFNγ (Clone XMG1.2,Cat#16–7311), anti-mouse IL-4(Clone 11B11, cat#16–7041), anti-mouse FOXP3 (clone FJK-16s, cat#17–5773) are from eBioscience. Anti-mouse CD4 (clone RM4–5, Cat#550954), anti-mouse CD25 (clone 7D4, Cat#558642), anti-mouse CD44 (Clone IM7, Cat#559250), anti-mouse CD62L (clone MEL-14, Cat#560507), anti-mouse IL-17 (clone TC11–18H10, Cat#559502), anti-mouse IFNγ (Clone XMG1.2, Cat#561040, for staining) and anti-CD90.1 (Thy1.1) (clone OX-7, cat#561406) were from BD Bioscience.
Anti mouse foxp3 clone fjk 16s
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse FOXP3 (clone FJK-16s) is a monoclonal antibody used for the detection of the transcription factor FOXP3 in mouse cells. FOXP3 is a key regulator of the development and function of regulatory T cells. This antibody can be used in flow cytometry and other immunological assays to identify and analyze FOXP3-expressing cells.
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Lab products found in correlation
Anti mouse ifn γ clone xmg1, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Anti mouse il 17 clone tc11 18h10, by BD (1 mentions)
Anti idh2, by Abcam (1 mentions)
Anti idh1, by Cell Signaling Technology (1 mentions)
Anti mouse cd3 clone 145 2c11, by Thermo Fisher Scientific (1 mentions)
Anti mouse ifnγ clone xmg1, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd44 clone im7, by BD (1 mentions)
Anti mouse cd62l clone mel 14, by BD (1 mentions)
Anti mouse cd28 clone 37, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4 clone rm4 5, by BD (1 mentions)
Anti hif 1α, by Novus Biologicals (1 mentions)
Anti mouse thy1.1 clone ox 7, by BD (1 mentions)
Anti mouse cd4 clone gk1, by BD (1 mentions)
Anti mouse cd25 clone pc61, by BD (1 mentions)
Anti mouse pd 1 clone j43, by Thermo Fisher Scientific (1 mentions)
Lsr 2, by BD (1 mentions)
Cd25 pe, by Thermo Fisher Scientific (1 mentions)
Cd103 percp cy5, by BioLegend (1 mentions)
Cellquest software, by BD (1 mentions)
7 protocols using anti mouse foxp3 clone fjk 16s
1
Murine Immune Cell Phenotyping
2
Multiparameter Immune Cell Phenotyping
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Cell surface staining was performed according to standard procedures using antibodies against anti-mouse CD4 (clone GK1.5; BD Pharmingen), anti-mouse Thy1.1 (clone OX-7; BD Pharmingen), anti-mouse Thy1.2 (clone 53-2.1; BD Pharmingen), anti-mouse LAG-3 (clone C9B7W; BD Pharmingen), anti-mouse TNFR2 (clone TR75-89; BD Pharmingen), anti-mouse CD25 (clone PC61; BD Pharmingen), anti-mouse CTLA-4 (clone UC10-F10-11; BD Pharmingen), anti-mouse PD-1 (clone J43; eBioscience), anti-mouse GARP (clone YGIC86; eBioscience), anti-mouse CD38 (clone 90; eBioscience), anti-mouse GITR (clone DTA-1; eBioscience), anti-mouse neuropilin-1 (clone 3DS304M; eBioscience), and anti-mouse CD73 (cloneTY/11.8; Biolegend). Foxp3 was detected using the GFP reporter or by intracellular cytokine staining using a anti-mouse Foxp3 (clone FJK-16 s; eBioscience) antibody. All flow cytometry was performed on a BD LSRII or BD FACSCalibur and analyzed using FlowJo (TreeStar). Surface stain was performed by incubating cells with antibody cocktail mix for 20 minutes at 4°C. For intracellular cytokine Foxp3 staining, cells were first stained for surface receptors, fixed in 4% formyl saline, permeabilized (0.5% BSA, 0.1% Triton, and 2 mM EDTA in PBS) for 45 minutes at room temperature. Cells were incubated overnight with the anti-cytokine antibodies and analyzed by flow cytometry.
3
Quantifying Immune Cell Profiles in Tumor Tissue
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Tumor tissue was freshly taken from mice and processed for IHC. IHC was performed on formaldehyde-fixed and paraffin-embedded 3–4 µm tissue sections, using rabbit monoclonal anti-mouse PD-1 (clone EPR20665, Abcam, USA), anti-mouse CD4 (clone EPR19514, Abcam, USA), anti-mouse CD8 (clone EPR21769, Abcam, USA) and anti-mouse FOXP3(clone FJK-16s, eBioscience). All images were examined by two experienced pathologists independently. The slides were then scanned into high-resolution images using Aperio Versa 200 (Aperio, Vista, California, USA). The images were then visualized and the percent of CD4+, CD8+FOXP3+ and PD1+ cells (eg, number of positive cells/total number of cells) were quantified using the software Aperio Image Scope (Aperio).
4
Murine Lymphocyte Phenotyping by Flow Cytometry
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Hepatic mononuclear cells (HMNCs) and splenocytes were isolated as previously described [18 (link)]. Single-cell suspensions of HMNCs and splenocytes were labelled with fluorescent conjugated antibodies against mouse CD4-FITC, CD25-PE (eBioscience), CD62L-PE Cy7, CTLA-4-PE (BD Pharmingen), as well as CD103-PerCP-Cy5.5 (Biolegend). Nonspecific binding was blocked with staining buffer supplemented with 2% mouse serum and anti-CD16/anti-CD32. For intracellular staining of Foxp3, mouse regulatory T cell staining Kit (eBioscience) was used. Cells were labeled with surface antibody, fixed and permeabilized with freshly prepared Fixation/Permeabilization working solution according to the manufacturer’s instructions. After permeabilization, cells were further labeled with anti-mouse foxp3 (clone FJK-16s) (eBioscience). After incubation and resuspension, HMNCs and splenocytes were evaluated by flow cytometry, and the data were analyzed using Cell Quest software (Becton Dickinson).
5
Multicolor Flow Cytometry Profiling
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Single-cell suspensions from spleen, peritoneal lavage, uterus, blood, inguinal (ILN) and para-aortic lymph nodes (PLN) were obtained and stained for cell surface markers for 30 min at 4 °C. The following anti-mouse fluorescently labeled antibodies were used: CD19 (clone 1D3) and CD4 (clone RM4-4; both from BD Biosciences, Germany), CD25 (clone 3C7), IgM (clone RMM-1), IgD (clone 11-26 c.2a) and B220 (clone RA3-6B2; all from Biolegend, San Diego, CA, USA). For detection of the intracellular expression of Foxp3, cell suspensions were fixed ON using Fix and Perm (ebioscience, Germany) and stained with anti-mouse Foxp3 (clone FJK-16s; ebioscience) for 30 min at 4 °C. Measurements were performed with a FACSCalibur and analyzed with CellQuestPro software (BD Biosciences).
6
Immune Cell Profiling of Psoriatic Skin
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On day seven, the ears of TPA-induced psoriasis mice were collected and finely ground. The cells were then soaked in media containing Liberase (Roche) and incubated for 1 h and 30 min. The cells were filtered using a 70 μm Nylon cell filter, centrifuged at 400 × g for 8 min, and red blood cells were removed with ammonium-Chloride-Potassium (ACK) lysing buffer and washed with PBS (Lorenz and von Stebut, 2014 (link)). The separated cells were stimulated for 4 h using phorbol-12-myristate-acetate (PMA), ionomycin, and a Golgi plug kit (BD Pharmingen). A viability staining kit (Invitrogen) was used to distinguish dead cells. Tregs and cytokines were expressed as anti-mouse CD4 (clone RM4-5, Biolegend), anti-mouse IFN-γ (clone XMG1.2, BD Pharmagen), anti-mouse Foxp3 (clone FJK-16s, eBioscience), anti-mouse IL-10 (clone JES5-16E3, BD Pharmagen), anti-mouse IL-17A (clone TC11-18H10, BD Pharmagen). In the FACS analysis, cells were stained with fluorescent dyes and monoclonal antibodies, and dead cells were excluded. Each cell was classified according to the fluorophore of the antibody, and the gate in the analysis was set using less than 0.5% isotype control. The FACS analysis was performed using a software from the manufacturer Attune NxT Flow Cytometer (Invitrogen).
7
Therapeutic Antibody and Immune Marker Analysis
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Therapeutic antibodies were as follows: anti-CEACAM1 mAb (Clone MAb-CC1; Cat. #: 134504; Biolegend, Santa Cruz, CA, USA) and Mouse IgG1 isotype control (Clone MOPC-21; Cat. #: 400153; Biolegend). Antibodies for flow cytometry and immunohistochemistry were as follows: anti-mouse CD3e (Clone 145-2C11; Cat. #: 15–0031-82; eBioscience, San Diego, CA, USA), anti-mouse CD4 (Clone GK1.5; Cat. #: 12–0041-82; eBioscience), anti-mouse CD8a (Clone 53–6.7; Cat. #: 11–0081-82; eBioscience), anti-mouse CD25 (Clone PC61.5; Cat. #: 25–0251-82; eBioscience), anti-mouse Foxp3 (Clone FJK-16 s; Cat. #: 11–5773-82; eBioscience), anti-mouse CEACAM1 (Clone 723629; Cat. #: MA5-24338; eBioscience), anti- human CD3 (Clone OKT3; Cat. #: 14–0037-82; eBioscience), anti- human CD4 (Clone RPA-T4; Cat. #: 11–0049-42; eBioscience), anti-mouse CD8a (Clone RPA-T8; Cat. #: 12–0088-42; eBioscience), anti-human CEACAM1 (Clone YTH71.3; Cat. #: MA5-17003; eBioscience),anti-mouse ki-67(SAB5700770; Sigma-Aldrich) and In Situ Cell Death Detection Kit (11684817910;Roche).
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