Fixation permeabilization solution kit

Manufactured by BD

Sourced in United States, Germany

The Fixation/Permeabilization Solution Kit is a laboratory product designed to prepare biological samples for analysis. The kit contains solutions for fixing and permeabilizing cells, which are essential steps in various experimental procedures, such as immunofluorescence staining and flow cytometry. The kit provides the necessary tools to prepare samples for further investigation, but it does not include instructions for specific applications or interpretations of the results.

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Lab products found in correlation

Golgiplug, by BD (17 mentions) Golgistop, by BD (16 mentions) Ionomycin, by Merck Group (15 mentions) Facscanto 2, by BD (15 mentions) Lsrfortessa, by BD (14 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (12 mentions) Lsr 2, by BD (10 mentions) Facsdiva software, by BD (10 mentions) Brefeldin a, by BioLegend (9 mentions) Phorbol myristate acetate (pma), by Merck Group (9 mentions) Facscalibur, by BD (9 mentions) Lsrii flow cytometer, by BD (9 mentions) Lsrfortessa flow cytometer, by BD (8 mentions) Brefeldin a, by Merck Group (7 mentions) Brefeldin a, by BD (6 mentions) Facscanto 2 flow cytometer, by BD (6 mentions) Perm wash buffer, by BD (6 mentions) Collagenase 4, by Merck Group (5 mentions) Rbc lysis buffer, by BD (5 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (5 mentions)

312 protocols using fixation permeabilization solution kit

Multiparametric Flow Cytometry Analysis of Immune Cell Phenotypes and Metabolic Regulators

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The cell surface markers and cytokines were analyzed by flow cytometry, as described previously [30 (link),31 (link),32 (link)]. Living cells were stained with the following antibodies in PBS containing 0.1% (weight/volume) bovine serum albumin and 0.1% NaN₃ for 30 min on ice. The following antibodies were obtained from BD Biosciences (Lake Franklin, NJ, USA): anti-CD4 (GK1.5), anti-B220 (RA36B2), anti-CXCR5 (2G8), anti-T- and B-cell activation antigen (GL-7), anti-CD95 (Jo2), anti-CD138 (281-2), and anti-IgD (11-26c.2a). The following antibodies were obtained from eBioscience (Thermo Fisher): anti-PD-1 (J43), anti-CXCR5 (SPRCL5), and anti-IL-21 (FFA21). To detect cytokine secretion, cells were stimulated with phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich) and ionomycin (PeproTech-BioGems, NJ, USA) for 5 hours. The cells were fixed using a Fixation/Permeabilization Solution Kit (BD Biosciences). To detect the metabolism-related regulators, we used anti-Glut1 (EPR3915) and anti-SDHα (EPR9043B) antibodies. Cells were fixed with a Fixation/Permeabilization Solution Kit (BD Biosciences) and were intracellularly stained for both molecules. All flow cytometry data were obtained with ACEA NovoCyte (ACEA Biosciences, Inc., San Diego, CA, USA), and the data were analyzed with NovoExpress (TreeStar, San Carlos, CA).

Dong L., He Y., Zhou S., Cao Y., Li Y., Bi Y, & Liu G. (2019). HIF1α-Dependent Metabolic Signals Control the Differentiation of Follicular Helper T Cells. Cells, 8(11), 1450.

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Flow Cytometry Immune Cell Profiling

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Cells were harvested from the tissues and pre-incubated with the anti-FcγR mAb and normal mouse serum to block nonspecific antibody binding for 15 min at 4C. Surface staining was performed in FACS buffer (PBS with 1% heat-inactivated FBS) for 25 min at 4C using the indicated surface Abs. Dead cells were excluded by DAPI staining and doublet events were eliminated by comparing the FCS-W and FCS-H to the SSC-W and SSC-H. IL-4 intracellular staining was performed using the BD Fixation/Permeabilization Solution Kit (BD Biosciences). Intracellular staining of PLZF was performed by fixing cells in 4% paraformaldehyde in PBS for 30 min at 4C. Cells were then permeabilized for 15 min at 4C in commercially-available Permeabilization Buffer (Invitrogen), and incubated with anti-PLZF antibody in Permeabilization Buffer for 1 hour at 4C. Samples were washed 3X with Permeabilization Buffer and 1X with FACS buffer prior to analysis. Events were acquired on an LSRII cytometer (BD Biosciences) and the data were analyzed with the FlowJo software.

Zhang S., Vieth J.A., Krzyzanowska A., Henry E.K., Denzin L.K., Siracusa M.C, & Sant’Angelo D.B. (2019). The Transcription Factor PLZF is Necessary for the Development and Function of Mouse Basophils. Journal of immunology (Baltimore, Md. : 1950), 203(5), 1230-1241.

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Vaccine Formulations Enhance Antigen-Specific T Cells

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Mice (C57BL/6, n = 5 in each group from two pooled experiments) were injected
intravenously with different vaccine formulations, such as multi-LP/α-GalCer
+ TRP2-mRNA, multi-LP/TRP2-mRNA, multi-LP/TRP2-mRNA + free α-GalCer,
free α-GalCer + TRP2-mRNA, and PBS as a negative control. The
vaccine formulations were prepared using 10 μg of TRP2-mRNA
and 0.5 μg of α-GalCer. Blood was collected from vaccinated
mice by saphenous bleeding and erythrocytes were removed by addition
of ACK lysis buffer (Quality Biological). peripheral blood mononuclear
cells (PBMCs) were stimulated with 1 μg/mL of TRP2-peptide (SVYDFFVWL)
for 6 h in the presence of a protein transport inhibitor, Brefeldin
A (BD GolgiPlug). After incubation, cells were washed and stained
with Abs for surface markers-TCRβ, CD8, and V450/viability dye.
Cells were then washed, fixed, and permeabilized with a BD Fixation/Permeabilization
Solution Kit and stained with anti-IFN-γ. The cells were washed,
resuspended in FACS buffer (1× PBS and 0.5% BSA), and analyzed
by flow cytometry (BD Fortessa X-20).
SVYDFFVWL-specific tetramer
(MBL International) staining was carried out by incubation at room
temperature for 10 min in FACS buffer. After washing, PBMCs were stained
with anti-TCRβ, anti-CD8 clone KT15, and V450/viability for
30 min at 4 °C in FACS buffer. Then, the samples were analyzed
by flow cytometry (BD Fortessa X-20).

Guevara M.L., Jilesen Z., Stojdl D, & Persano S. (2019). Codelivery of mRNA with α-Galactosylceramide Using a New Lipopolyplex Formulation Induces a Strong Antitumor Response upon Intravenous Administration. ACS Omega, 4(8), 13015-13026.

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Flow Cytometric Analysis and FACS Sorting

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For flow cytometric analyses and FACS sorts, lineage-depleted, CD4⁺ T cell enriched or unfractionated cells were stained in PBS 2% FCS for 20 min with corresponding antibodies and washed. For Y-Ae antibody conjugated with biotin, cells were washed and incubated for another 20 minutes with Streptavidin-PE (ThermoFisher). For intracellular cytokine staining, cells were stimulated for 4h at 37°C with the Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience). After surface staining, cells were fixed, permeabilized and stained using the BD Fixation/Permeabilization Solution Kit (BD Biosciences) according to manufacturer’s instructions. Finally, cells were filtered through a 35-40μM filter and acquired by a flow cytometer (LSR II or LSRFortessa, Becton Dickinson) or cell sorter (FACSAria II or FACSAria Fusion, Becton Dickinson) for analysis or sort, respectively. Common gating strategies used in this study to define populations are depicted in Figure S6 and S7.

Hernández-Malmierca P., Vonficht D., Schnell A., Uckelmann H.J., Bollhagen A., Mahmoud M.A., Landua S.L., van der Salm E., Trautmann C.L., Raffel S., Grünschläger F., Lutz R., Ghosh M., Renders S., Correia N., Donato E., Dixon K.O., Hirche C., Andresen C., Robens C., Werner P.S., Boch T., Eisel D., Osen W., Pilz F., Przybylla A., Klein C., Buchholz F., Milsom M.D., Essers M.A., Eichmüller S.B., Hofmann W.K., Nowak D., Hübschmann D., Hundemer M., Thiede C., Bullinger L., Müller-Tidow C., Armstrong S.A., Trumpp A., Kuchroo V.K, & Haas S. (2022). Antigen presentation safeguards the integrity of the hematopoietic stem cell pool. Cell stem cell, 29(5), 760-775.e10.

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Single-cell Multiparametric Immunophenotyping

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Single-cell suspensions were stained with antibodies against cell-surface antigens while in Hanks’ Balanced Salt Solution containing 1% bovine serum albumin and 2 mM EDTA. To detect intracellular cytokines and transcription factors, cells were then fixed, permeabilized, and intracellular stained using BD Fixation/Permeabilization Solution Kit (BD Biosciences). Antibodies were purchased from BD Biosciences, eBiosciences, or BioLegend. Flow cytometry was performed using BD LSR II and data analyzed using FlowJo version 9.9.6.

Chen E.L., Lee C.R., Thompson P.K., Wiest D.L., Anderson M.K, & Zúñiga-Pflücker J.C. (2021). Ontogenic timing, T cell receptor signal strength, and Notch signaling direct γδ T cell functional differentiation in vivo. Cell reports, 35(10), 109227.

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Comprehensive T cell Immunophenotyping

Cited 1 time

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Immunophenotype of T cells was performed using standard staining and flow cytometry techniques as described before^{41 (link),42 (link)}. Briefly, combinations of fluorophore-conjugated anti-human monoclonal antibodies specific for CD3 (BioLegend, OKT3, Cat#317344, 1:500), CD4 (BioLegend, OKT4, Cat#317438, 1:500), CD8 (BioLegend, SK1, Cat#344724, 1:500), CCR7 (BioLegend, G043H7, Cat#353214, 1:500), CD45RO (BioLegend, UCHL1, Cat#304244, 1:500), PD1 (eBioscience, EBIOJ105, Cat#12-2799-42, 1:500), TIM3 (BioLegend, F38-2E2, Cat#345006, 1:500), LAG3 (eBioscience, 3DS223H, Cat#15-2239-42, 1:500) were used to label T cells in flow cytometry staining buffer for 30 min on ice after Fc blocking, followed by intracellular cytokine staining with the BD Fixation/Permeabilization Solution Kit. Data were acquired on LSRFortessa (BD Biosciences) and analyzed with FlowJo software (Treestar). Cytokine antibodies included IFN-γ (eBioscience, B27, Cat#MHCIFG04, 1:500), IL9 (BioLegend, MH9A4, Cat#507614, 1:500), IL2 (BioLegend, MQ1-17H12, Cat#500342, 1:500), GrzB (BD Biosciences, GB11, Cat#561142, 1:500), or annexin V (BD Bioscience, Cat#550475, 1:50).

Liu L., Bi E., Ma X., Xiong W., Qian J., Ye L., Su P., Wang Q., Xiao L., Yang M., Lu Y, & Yi Q. (2020). Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9. Nature Communications, 11, 5902.

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Multiparametric Analysis of Immune Cells

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Mouse thymus, spleen, and lymph node (LN) were processed into single cell suspensions, and analyzed by flow cytometry. Thymic epithelial cells (TECs) were isolated as previously described (34 (link)). Dead cells were stained by a fixable viability dye conjugated to eFluor780 (eBioscience). Fixation and intracellular/intranuclear staining were performed using the eBioscience Foxp3 staining kit or BD Fixation/Permeabilization Solution Kit. The following antibodies were purchased from BD biosciences: TCRβ (H57–597), TCRγδ (GL-3) and EpCAM (G8.8). The following antibodies were purchased from eBioscience: CD3 (145–2C11), CD4 (GK1.5), CD11c (N418), CD25 (PC61.5), CD45R (B220, RA3–6B2), MHCII (M5/114) and Ki67 (SolA15). The following antibodies were purchased from BioLegend: CD8 (53–6.7), CD11b (M1/70), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), CD122 (TM-β1), Gr-1 (RB6–8C5), Ly-51 (6C3), NK-1.1 (PK136) and TER-119 (TER-119). UEA-1 was purchased from Vector Laboratories. Data were collected using a BD LSR II flow cytometer and were analyzed using FlowJo V10 (Treestar Inc.). The TaqMan Gene Expression Assay (ID: Mm00502000_m1, Thermo Fisher Scientific) for quantification of Klotho gene expression.

Xing Y., Smith M.J., Goetz C.A., McElmurry R.T., Parker S.L., Min D., Hollander G.A., Weinberg K.I., Tolar J., Stefanski H.E, & Blazar B.R. (2018). Thymic epithelial cell support of thymopoiesis does not require Klotho. Journal of immunology (Baltimore, Md. : 1950), 201(11), 3320-3328.

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Evaluating antigen-specific T-cell responses

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Cryopreserved patients’ PBMCs from the same patients of different time points were thawed and cultured at the same time. PBMCs were cultured for 10 days in STEMCELL™-XF T cell expansion medium with 50IU/ml of IL-2, in the presence of 15-mer, overlapping by 11 amino acids peptide pools derived from human NY-ESO-1, MAGE-A4, or PRAME proteins (JPT, Berlin, Germany), or without peptide as a negative control. Half medium change was performed every 3–4 days. On day 10, cells were re-stimulated with the same stimulant as in culture for 6 hours, with the presence of brefeldin A (eBioscience) for the last 5 hours. Single peptide concentrations used in culture and restimulation were both 1ug/ml. Staining of CD4-BV421, CD8-APC-Cy7 and eBioscience fixable viability dye eFluor 506 was performed prior to permeabilization and fixation using BD Fixation/Permeabilization Solution Kit. IFNγ-FITC and TNF-PE-Cy7 were stained intracellularly for 30 min prior to flow cytometry analysis.

Zhang S., Kohli K., Black R.G., Yao L., Spadinger S.M., He Q., Pillarisetty V.G., Cranmer L.D., Van Tine B.A., Yee C., Pierce R.H., Riddell S.R., Jones R.L, & Pollack S.M. (2019). Systemic Interferon-γ Increases MHC Class I Expression and T-cell Infiltration in Cold Tumors: Results of a Phase 0 Clinical Trial. Cancer immunology research, 7(8), 1237-1243.

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Intracellular Cytokine Staining Assay

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Cells were stimulated with phorbol myristate acetate and ionomycin, and treated with brefeldin A (Biolegend) for 4 hours before staining for intracellular cytokines with the BD Fixation/Permeabilization Solution Kit. Results were then acquired using BD Calibur, BD Fortessa or Miltenyi MACSQuant systems. Data were analyzed with FlowJo_V10 software (TreeStar).

Ma X., Bi E., Lu Y., Su P., Huang C., Liu L., Wang Q., Yang M., Kalady M.F., Qian J., Zhang A., Gupte A.A., Hamilton D.J., Zheng C, & Yi Q. (2019). Cholesterol induces CD8+ T-cell exhaustion in the tumor microenvironment. Cell metabolism, 30(1), 143-156.e5.

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Age-Dependent LPS-Induced IL-6 Secretion

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Young (3 mo old) and old (24 mo old) WT mice received an i.p. injection of LPS (1.5 mg/kg) and were sacrificed 3 h later. 5 × 10⁶ c-Kit⁺–enriched BM cells were isolated and cultured in 1 ml of SFEM with 100 U/ml penicillin, 100 µg/ml streptomycin, 50 ng/ml TPO, and 50 ng/ml SCF in a 24-well plate in the presence of secretion inhibitor Brefeldin A (BD Biosciences; 555029). After 4 h culture, cells were fixed and permeabilized using BD Fixation/Permeabilization Solution kit (554714). The level of IL-6 production in the HSC population was analyzed by FACS using IL-6 antibody (BD Biosciences; 554401).

Chen Z., Amro E.M., Becker F., Hölzer M., Rasa S.M., Njeru S.N., Han B., Di Sanzo S., Chen Y., Tang D., Tao S., Haenold R., Groth M., Romanov V.S., Kirkpatrick J.M., Kraus J.M., Kestler H.A., Marz M., Ori A., Neri F., Morita Y, & Rudolph K.L. (2019). Cohesin-mediated NF-κB signaling limits hematopoietic stem cell self-renewal in aging and inflammation. The Journal of Experimental Medicine, 216(1), 152-175.

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