Foxp3 fix perm solution

Manufactured by BioLegend

Sourced in United States

Foxp3 Fix/Perm solution is a reagent used for the intracellular staining and detection of the transcription factor Foxp3. It is designed to fix and permeabilize cells, allowing for the efficient labeling of intracellular proteins.

Automatically generated - may contain errors

Lab products found in correlation

10 protocols using foxp3 fix perm solution

Foxp3 Expression Analysis of Regulatory T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Assessment of intracellular Foxp3 expression was used to identify regulatory T cells. Activated CD4⁺ T cells were prepared as described above. These were stained with antibodies specific to CD4, Vγ2 and Thy1.2 prior to resuspension in 200 μl of Foxp3 Fix/Perm solution (Biolegend) and incubation in the dark for 20 min. Cells were washed once with FACS buffer, followed by washing with Foxp3 Fix/Perm buffer. Supernatant was discarded completely, and cells resuspended in 200 μl Foxp3 Perm buffer (Biolegend). Cells were incubated in the dark for a further 15 min and then centrifuged to remove supernatant. The pellet was resuspended in 20 μl anti-Foxp3-PE conjugate (Biolegend) with incubation in the dark for 30 min. Cells were washed twice with FACS buffer and Foxp3 expression determined by flow cytometric analysis.

Petvises S., Periasamy P, & O’Neill H.C. (2018). MCSF drives regulatory DC development in stromal co-cultures supporting hematopoiesis. BMC Immunology, 19, 21.

+ Open protocol

+ Expand

Foxp3+ Regulatory T-Cell Immunophenotyping

Check if the same lab product or an alternative is used in the 5 most similar protocols

Prediluted antibodies were prepared for staining of cell surface markers. For intracellular staining, cells were permeabilized in Foxp3 Fix/Perm solution (BioLegend) and incubated with anti-Foxp3 antibodies diluted in Foxp3 Perm buffer (BioLegend). The antibodies used: anti-CD3-PerCP (clone 145-2C11, BioLegend), anti-CD4-Pacific blue (clone RM4-5, BioLegend), anti-Foxp3-Alexa Fluor 488 (clone FJK-16s, Thermo Fisher Scientific), and anti-Nrp-1-APC (clone 3DS304M, Thermo Fisher Scientific). FACSCanto II (BD Biosciences) and FlowJo software (BD Biosciences) were used to collect and analyze the data.

Chi J.N., Yang J.Y., Hsueh C.H., Tsai C.Y., Chuang H.C, & Tan T.H. (2022). MAP4K3/GLK inhibits Treg differentiation by direct phosphorylating IKKβ and inducing IKKβ-mediated FoxO1 nuclear export and Foxp3 downregulation. Theranostics, 12(13), 5744-5760.

+ Open protocol

+ Expand

Comprehensive Immune Cell Isolation and Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Spleens and liver-draining lymph nodes were collected from humanely euthanized mice between 8–12 weeks of age and homogenized through a 70 μm cell strainer, then further suspended using a 27-gauge needle. Liver non-parenchymal cells were isolated by two-step perfusion as described in “primary hepatocyte isolation and culture”. Hepatocytes were removed from the cell suspension by sequential centrifugation at 50xg; the supernatants containing NPCs were centrifuged at 300xg for 5 minutes to pellet the cells. Red blood cells were removed from NPCs, splenocytes, and lymph node suspensions by incubating in ACK lysis buffer for 5 minutes at room temperature. Cleared cells were pelleted by centrifugation at 500 g for 5 minutes, then washed twice in phosphate-buffered saline (PBS).
Surface marker staining was performed as follows: First, viability staining was performed for 20 minutes on ice in PBS with Tonbo Ghost Dye UV450, with all samples protected from light through this and subsequent steps. Conjugated antibodies were added and cells were incubated 15–20 minutes on ice. See Supplemental File 1 for antibody panels used. Cells were washed 3 times in staining buffer (PBS with 5% FBS and 0.1% sodium azide).
Intracellular staining was performed using the Foxp3 Fix/Perm solution (BioLegend) according to manufacturer’s instructions.

Hanley K.L., Liang Y., Wang G., Lin X., Yang M., Karin M., Fu W, & Feng G.S. (2022). Concurrent Disruption of Ras/MAPK and NF-κB Pathways Induces Circadian Deregulation and Hepatocarcinogenesis. Molecular cancer research : MCR, 20(3), 337-349.

+ Open protocol

+ Expand

Multiparameter Analysis of Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin or with 5 μg/ml αCD3 mAb as specified in figure legends in the presence of brefeldin A (BioLegend). Cells were collected, washed twice in FACS buffer, and stained for surface markers on ice for 20 min. Cells were washed twice of excess Ab and fixed with FOXP3 Fix/Perm Solution (BioLegend) at room temperature for 20 min. Cells were washed in FACS buffer prior to washing and suspension in FOXP3 Perm Buffer for 15 min. Permeabilized cells were incubated with intracellular Abs for 30 min. Excess Ab was washed off twice before suspension in FACS buffer. Surface markers include mouse α-mouse CD3-PeCy7 (145–2C11), α-mouse CD25-APC/Cy7 (PC61), α-mouse CD4-PerCP/Cy5.5 (GK1.5), α-mouse CD11b–Brilliant Violet 510 (M1/70), α-mouse CD11c–Pacific Blue (N418), α-mouse CD45.2– Brilliant Violet 605 (104), α-mouse F4/80-APC (BM8), α-mouse Ly-6C–Alexa Fluor 700 (HK1.4), and α-mouse CD115-PerCP/Cy5.5 (AFS98). Intracellular marker included mouse α-mouse IL-10–APC (JES5–16E3). All mouse Abs were purchased from BioLegend. Mouse samples were run on a BD LSR II or BD LSRFortessa (BD Biosciences, Franklin Lakes, NJ) and analyzed using FlowJo (Ashland, OR).

Johnson B.M., Uchimura T., Gallovic M.D., Thamilarasan M., Chou W.C., Gibson S.A., Deng M., Tam J.W., Batty C.J., Williams J., Matsushima G.K., Bachelder E.M., Ainslie K.M., Markovic-Plese S, & Ting J.P. (2021). STING Agonist Mitigates Experimental Autoimmune Encephalomyelitis by Stimulating Type I IFN–Dependent and –Independent Immune-Regulatory Pathways. Journal of immunology (Baltimore, Md. : 1950), 206(9), 2015-2028.

+ Open protocol

+ Expand

Prostate Tissue Dissociation and Myeloid Cell Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Prostate tissue was subjected to single cell dissociation using the MACS Mouse Tumor Dissociation Kit protocol and gentleMACS Dissociator (Miltenyi). Suspended cells were blocked with rat serum (012–000-120, Jackson ImmunoResearch), stained with FVS570 viability dye (1 ul/ml, 564995, BD Biosciences) in the dark for 15 minutes at room temperature. Samples were washed with PBS and incubated with Myeloid extracellular antibody panel (Supplementary Table S1) or corresponding isotype panels diluted in Brilliant Stain Buffer (566349, BD Biosciences) in the dark for 30 minutes at 4°C. Cells were washed with FACS buffer (1x PBS, 1% BSA, 2mM EDTA), fixed with 1x Fixation Buffer (420801, BioLegend) in the dark for 20 minutes at room temperature, and stored overnight in FACS buffer at 4°C. Samples were incubated in 1x FoxP3 Fix/Perm Solution (421401, BioLegend) in the dark for 20 minutes at room temperature and washed with 1x FoxP3 Perm Buffer (421402, BioLegend). Cells were resuspended with Myeloid intracellular antibody panel (Supplementary Table S1) or corresponding isotype panels diluted in FACS buffer in the dark for 30 minutes at room temperature under gentle agitation. Cell suspensions were washed with FACS buffer and analyzed with a Gallios flow cytometer (Beckman Coulter Life Sciences). Macrophages were defined as FVS570⁻CD45⁺CD11b⁺F4/80⁺CD68⁺ cells.

de Groot A.E., Myers K.V., Krueger T.E., Kiemen A.L., Nagy N.H., Brame A., Torres V.E., Zhang Z., Trabzonlu L., Brennen W.N., Wirtz D., De Marzo A.M., Amend S.R, & Pienta K.J. (2021). Characterization of tumor-associated macrophages in prostate cancer transgenic mouse models. The Prostate, 81(10), 629-647.

+ Open protocol

+ Expand

Multiparameter Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Single cell suspensions were prepared from spleens and draining lymph nodes of WT and Mif^−/− mice, washed in PBS and blocked with normal mouse serum. Cells were incubated with fluorochrome conjugated antibodies against the cell surface markers: CD4, CD8, CD3, NK1.1, F4/80, CD11b, Ly6C and Ly6G (Biolegend, SanDiego, CA). For FoxP3 intracellular staining, surface-stained cells were fixed and permeabilized with FoxP3 Fix/Perm solution (Biolegend, SanDiego, CA), then stained with PE-conjugated anti-FoxP3 antibodies (Biolegend, SanDiego, CA). Cells were acquired on a BD FACS Calibur or BD FACS Aria (BD Biosciences, San Jose, CA) and analysis was performed using the FlowJo software (Tree Star, Inc., Ashland, OR).

Oghumu S., Knobloch T.J., Terrazas C., Varikuti S., Ahn-Jarvis J., Bollinger C.E., Iwenofu H., Weghorst C.M, & Satoskar A.R. (2016). Deletion of macrophage migration inhibitory factor inhibits murine oral carcinogenesis: Potential role for chronic pro-inflammatory immune mediators. International journal of cancer, 139(6), 1379-1390.

+ Open protocol

+ Expand

Immunophenotyping of Murine Lymphocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols

Thymocytes, splenocytes, lymph node cells, and bone-marrow-derived cells were harvested from 5-week-old mice. For surface staining, cells were washed by PBS and were stained with antibodies at room temperature for 1 h (anti-CD3, anti-CD4, anti-CD8, anti-B220, and anti-IgM antibodies). After incubation, cells were washed twice with PBS buffer to remove unbound antibodies. For intracellular Foxp3 staining, cells were permeabilized using Foxp3 Fix/Perm solution (BioLegend) and then incubated with diluted anti-Foxp3 antibodies in Foxp3 Perm buffer (BioLegend) at 4 °C for overnight. After incubation, cells were washed twice with Foxp3 Perm wash buffer (BioLegend) to remove unbound antibodies. For flow cytometry, data were acquired with a FACS CantoII (BD Biosciences) and analyzed with FlowJo (v10.8.1) analytical software.

Shih Y.C., Chen H.F., Wu C.Y., Ciou Y.R., Wang C.W., Chuang H.C, & Tan T.H. (2024). The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling. Nature Communications, 15, 532.

+ Open protocol

+ Expand

Multiparameter Analysis of Murine Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Blood, diluted in EDTA, and the spleen were harvested from mice at various time points. Isolated splenocytes were prepared by removing the spleen membrane and passing through a 70-μm filter. Splenocytes and blood cells were washed, and RBCs were lysed as previously described (32 (link)). Single cells were incubated with anti-CD16/32 (93; BioLegend, San Diego, CA) to block Fc receptors to prevent nonspecific Ab binding and then were stained with the following mAbs conjugated to FITC, PE, allophycocyanin, or PE-indotricarbocyanine: anti-mouse CD3 (17A2; BioLegend), anti-mouse CD8 (53-6.7; BioLegend), anti-mouse CD122 (TM-β1; BioLegend), anti-mouse CD4 (GK1.5; BioLegend), anti-mouse CD25 (PC61; BioLegend), and anti-mouse CD49b (HMa2; BioLegend) Abs. For intranuclear staining, the cells were fixed and permeabilized with Foxp3 Fix/Perm solution (BioLegend) and then stained with PE-conjugated anti-mouse Helios Ab (22F6; BioLegend). The cells were analyzed using FACSCalibur in conjunction with FlowJo analysis software (BD Bioscience, Tokyo, Japan) or Gallios in conjunction with Kaluza analysis software (Beckman Coulter, Brea, CA).

Morita N., Hoshi M., Tezuka H., Ando T., Yoshida S., Sato F., Yokoi H., Ito H, & Saito K. (2023). CD8+ Regulatory T Cells Induced by Lipopolysaccharide Improve Mouse Endotoxin Shock. ImmunoHorizons, 7(5), 353-363.

+ Open protocol

+ Expand

Melanoma Cell Proliferation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Melanoma cells were collected and 1 ml of 1× FOXP3 Fix/Perm solution (BioLegend, Inc., San Diego, CA, USA) was added to each for 20 min and then were stained with anti-Ki67 antibodies (1:20; cat. no. 350514; BioLegend, Inc.) for 30 min according to the manufacturer's instructions. Isotype IgG antibody (1:20; cat. no. 400141; BioLegend, Inc.) was used as a control. Treated samples were analyzed with a CytoFLEX cytometer and CytExpert software (version 2.0) (both from Beckman Coulter Life Sciences, College Park, MD, USA). The mean fluorescence intensity (MFI) of Ki67 was detected. MFI refers to the mean of the fluorescence intensity in the fluorescence channel. The experiments were performed in triplicate.

Zhou W.J., Wang H.Y., Zhang J., Dai H.Y., Yao Z.X., Zheng Z., Meng-Yan S, & Wu K. (2020). NEAT1/miR-200b-3p/SMAD2 axis promotes progression of melanoma. Aging (Albany NY), 12(22), 22759-22775.

+ Open protocol

+ Expand

Optimized Treg Immunophenotyping Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

For Treg staining assays, cryopreserved PBMNCs were thawed and washed three times with PBS. Staining for Tregs was performed according to the manufacturer's instructions. Briefly, 1×10 6 cells were incubated on ice for 20 min after staining with a 10-μL cocktail of PerCP-Cy5.5-conjugated CD4 (Becton Dickinson [BD] Biosciences, San Jose, USA) and phycoerythrin (PE)-conjugated CD25 (anti-IL-2R) (BD Biosciences, San Jose, USA). The cells were washed twice with PBS and then FOXP3 fix/perm solution (BioLegend, San Diego, CA) was added to each tube. The re-suspended cells were incubated in darkness for 20 minutes at room temperature. After washing twice with permeabilization buffer, the cells were stained with 5 μL Alexa Fluor 488-conjugated anti-human FOXP3 (BioLegend, San Diego, USA) or Alexa Fluor 488-conjugated mouse IgG1 κ isotype control antibody (BioLegend, San Diego, USA) as a negative control and incubated in darkness for 30 minutes [14] .

Nishimura T., Inaba Y., Nakazawa Y., Omata T., Akasaka M., Shirai I, & Ichikawa M. (2015). Reduction in peripheral regulatory T cell population in childhood ocular type myasthenia gravis. Brain & development, 37(8).

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!