Cytofix cytoperm fixation permeabilization

Manufactured by BD

Sourced in United States

BD Cytofix/Cytoperm™ Fixation/Permeabilization is a reagent system designed for the intracellular staining of cells. It provides simultaneous fixation and permeabilization, allowing for the detection of intracellular proteins and antigens.

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Lab products found in correlation

Fix perm, by BD (1 mentions) Interleukin 2 (il 2), by Merck Group (1 mentions) Interleukin 6 (il 6), by CellGenix (1 mentions) αcd3 αcd28 dynabeads, by Thermo Fisher Scientific (1 mentions) Anti ifny, by BD (1 mentions) Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions) Macsquant analyzer 10, by Miltenyi Biotec (1 mentions) Facsverse, by BD (1 mentions) Lsrii system, by BD (1 mentions) Anti cd3 fluorescein isothiocyanate, by BD (1 mentions) Anti cd8 phycoerythrin, by BD (1 mentions) Anti ifn γ apc, by BD (1 mentions) Lsr 2 cfi, by BD (1 mentions) Cd8a apc cy7, by BD (1 mentions) Cd4 bv711, by BD (1 mentions) Golgiplug, by BD (1 mentions) Live dead fixable dead cell stain, by Thermo Fisher Scientific (1 mentions) Phospho eif2α, by Cell Signaling Technology (1 mentions) Phospho perk, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Cytofix/Cytoperm Fixation/Permeabilization solution BD Cytofix/Cytoperm fixation and permeabilization kit Cytofix/Cytoperm Cell Permeabilization/Fixation Solution BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit with BD GolgiPlug Protein transport inhibitor containing Brefeldin A BD Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit Cytofix/Cytoperm Fixation/Permeabilization Buffer BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit BD Cytofix/Cytoperm Fixation/Permeabilization Kit BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit

7 protocols using cytofix cytoperm fixation permeabilization

Mycobacterial Viability Assay

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M. smegmatis (ATCC 21732) was subcultured in Middelbrook 7H9 broth (Fischer, BD part number 221832) and Tween 80 (Fischer, BP338-500). They were then grown in a 37 °C CO₂ incubator for a week and extracted during the log phase of their growth. The mycobacteria were then separated into three different groups: live mycobacteria, mycobacteria killed with 70% ethanol, and mycobacteria killed with BD Cytofix/Cytoperm^™ Fixation/Permeabilization (BD Perm/Fix; San Jose, California). Each mycobacterial variation was prepared to an optical density of 1, which is approximately 3 × 10⁸ cfu/ml.
A 10 μl inoculating loop was used to plate a sample of each variation of the mycobacteria on BD BBL Stacker plates (A7 agar; San Jose, California). Two plates were made for each variation. A sample of 20 ml was extracted with a variable pipette and placed on a microscope slide. The slide was then covered with a coverslip and sealed with clear nail polish. Five microscope slides were prepared for live mycobacteria, mycobacteria killed with 70% ethanol, and mycobacteria killed with BD Perm/Fix (totaling 15 sample slides for each experiment).

Wong C., Ha N.P., Pawlowski M.E., Graviss E.A, & Tkaczyk T.S. (2016). Differentiating between live and dead Mycobacterium smegmatis using autofluorescence. Tuberculosis (Edinburgh, Scotland), 101 Suppl, S119-S123.

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T Cell Proliferation and Activation in 2D and 3D Environments

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Human pan T cells were stained with CellTrace™ CFSE Cell Proliferation Kit (Invitrogen) to track cell proliferation by flowcytometry. To pre-activate T cells, pan T cells were stimulated overnight with αCD3/αCD28 Dynabeads (Gibco). The following day, T cells were harvested and re-plated into two-dimensional (2D) medium, 3D collagen matrices or 3D RGD-PIC hydrogels at varying cell densities as indicated. Alternatively, unstimulated T cells (1 × 10⁶ cells/ml) were mixed with αCD3/αCD28 Dynabeads (Gibco) or with IL-2 (90-100 IU/mL), PHA (phytohaemagglutinin, 1 μg/mL, Sigma Aldrich) and IL-6 (15 ng/mL, Cell Genix). Subsequently, cells were embedded within the RGD-PIC hydrogels or collagen matrices at varying densities to test in situ activation. Cells were recovered from the RGD-PIC gels and collagen gels and proliferation by CFSE dilution and activation were assessed by flowcytometry on a FACS Verse (BD Biosciences). Cell numbers were quantified using a MACSQuant Analyzer 10 (Miltenyi Biotec). Fixable Viability Dye eFluor® 780 (eBioscience) was used to exclude dead cells. Intracellular staining for interferon gamma (IFNy) was done using anti-IFNy (BD Biosciences) and the BD Cytofix/Cytoperm Fixation/Permeabilization.

Weiden J., Voerman D., Dölen Y., Das R.K., van Duffelen A., Hammink R., Eggermont L.J., Rowan A.E., Tel J, & Figdor C.G. (2018). Injectable Biomimetic Hydrogels as Tools for Efficient T Cell Expansion and Delivery. Frontiers in Immunology, 9, 2798.

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Apoptotic Pathway Analysis in Cells

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Cells were seeded at a density of 5×10⁵ cells/well in six-well culture plates. Following treatment with IC₅₀ concentrations of damnacanthal for 72 h, cells were harvested and fixed by BD Cytofix/Cytoperm™ Fixation/Permeabilization (BD Biosciences). The cells were stained with fluorochrome-conjugated monoclonal anti-mouse p53 and Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), XIAP (BD Biosciences) and ER-α (Abcam, Cambridge, MA, USA). The cells were washed twice with PBS to remove non-specific binding stained with secondary fluorochrome-conjugated monoclonal antibody mouse anti-p53, -Bcl2, -XIAP and -ER-α (Abcam). The cells were washed twice and analyzed by BD FACSCalibur multicolor flow cytometer (BD Biosciences).

AZIZ M.Y., OMAR A.R., SUBRAMANI T., YEAP S.K., HO W.Y., ISMAIL N.H., AHMAD S, & ALITHEEN N.B. (2014). Damnacanthal is a potent inducer of apoptosis with anticancer activity by stimulating p53 and p21 genes in MCF-7 breast cancer cells. Oncology Letters, 7(5), 1479-1484.

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Cytokine Production Analysis in PBMCs

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PBMCs were stimulated with phorbol myristate acetate (50 ng/mL) and ionomycin (1 µg/mL) in the presence of brefeldin A (10 µg/mL) for 6 h. Cells were stained with anti-CD3-fluorescein isothiocyanate (BD Pharmingen, San Jose, CA, USA; Clone UCHT1), anti-CD4–peridinin–chlorophyll–protein complex (BD Pharmingen, San Jose, CA, USA; Clone SK3), anti-CD8–phycoerythrin (BD Pharmingen, San Jose, CA, USA; Clone HIT8α) for 30 min in the dark at 4 ^∘C. Cells were then fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization (BD Biosciences, San Jose, CA, USA) and stained with anti-IL-9–allophycocyanin (R&D Systems, Minneapolis, MN, USA; Clone #623153). Flow cytometric analysis was performed using a BD LSR II System (BD Biosciences, San Jose, CA, USA).

Li Y., Li L., Zhang W, & Gao Y. (2023). Amphiregulin/epidermal growth factor receptor/hypoxia-inducible factor-1α pathway regulates T helper 9 and T cytotoxic 9 cell response in adult patients with infectious mononucleosis. Biomolecules and Biomedicine, 23(1), 63-72.

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Lung Immune Response to RBD Vaccination

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Left lungs from HD-Ad_RBD vaccinated and control mice were harvested and digested for 45 min at 37 °C in digestion buffer containing liberase 2 μg/ml and Type IV DNase I 25 units/ml. The lung infiltrated cells were cultured with purified RBD protein at 10 μg/ml for 12 h at 37 °C followed by a 6 h treatment with GolgiPlug (BD 555028). After blocking with FcγIII and FcγII receptors antibodies (BD Pharmingen, 553142), cells were stained with live/dead fixable cell stain (Invitrogen 34955), CD44 BV510, CD4 BV711, and CD8a APC-Cy™7 (BD) antibodies. Stained cells were fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization (BD 555028), and then intracellularly stained with anti-IFN-γ APC (BD). Cells were analysed on a Becton Dickinson LSR II CFI (SickKids Flow Cytometry Facility), using Flowjo × 10.0 software.

Cao H., Mai J., Zhou Z., Li Z., Duan R., Watt J., Chen Z., Bandara R.A., Li M., Ahn S.K., Poon B., Christie-Holmes N., Gray-Owen S.D., Banerjee A., Mossman K., Kozak R., Mubareka S., Rini J.M., Hu J, & Liu J. (2021). Intranasal HD-Ad vaccine protects the upper and lower respiratory tracts of hACE2 mice against SARS-CoV-2. Cell & Bioscience, 11, 202.

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Multiparameter Flow Cytometry of Cellular Stress Responses

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At first, cells were incubated with 100 µl of CD16/32 Fc block diluted 1/50 in PBS for 15 min at 4°C. After wash, cells were stained in PBS with CD45.2-APC/Cy7, CD45.1-BV421, CD11b-BUV737, Ly- 6G/C-PE/Cy7 mouse antibodies (Table 7), and a LIVE/DEAD™ fixable dead cell stain (Thermo Fisher Scientific; Cat# L34961) in the dark for 30 min at RT. Then cells were washed, pelleted, and fixed with Cytofix/Cytoperm Fixation & Permeabilization (BD Biosciences). Samples were washed twice with 1X BD Perm/Wash™ Buffer (BD Biosciences) + 2% FBS (WashB), being pelleted at 1,000 g x 5 min at 4°C. Then cells were incubated overnight at 4°C with the following mouse primary antibodies: ATF-4 (Cell Signaling Technology), AFT-6 (Cell Signaling Technology), BiP (Cell Signaling Technology), CHOP (Cell Signaling Technology), phospho-eIF2α (Cell Signaling Technology), or phospho-PERK (Thermo Fisher Scientific). The next day, samples were washed four times and pelleted. Cells were incubated with a donkey anti-rabbit AF488 secondary antibody (Thermo fisher Scientific) for 1-hour at 4°C, after which cells were washed four times and pelleted. Samples were then resuspended in FACS buffer and analyzed on an LSRII (BD Biosciences).

Sohn J., Milosevic J., Brouse T., Aziz N., Elkhoury J., Wang S., Hauschild A., van Gastel N., Cetinbas M., Tufa S.F., Keene D.R., Sadreyev R.I., Pu W.T, & Sykes D.B. (2022). A new murine model of Barth syndrome neutropenia links TAFAZZIN deficiency to increased ER stress-induced apoptosis. Blood Advances, 6(8), 2557-2577.

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Flow Cytometric Analysis of SARS-CoV-2-specific T Cells

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For extracellular staining, the TCR-T cells were stained with Live/Dead Fixable Yellow Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA) for 10 min at room temperature and then stained with anti-human CD3 (clone SK7) and CD8 (clone SK1) antibodies for 20 min at 4°C. To detect the expression of the introduced SARS-CoV-2-specific TCRs, the electroporated T cells were stained with TCR V β antibodies or Pentamer for 20 mins at 4°C. For intracellular staining, the cells were fixed and permeabilized using the Cytofix/Cytoperm fixation/permeabilization (BD Biosciences, San Jose, CA) buffer following the manufacturer's protocols. Intracellular cytokine staining was performed with anti-human IL-2, IFNg and TNFa (BD Biosciences) antibodies for 30 min at room temperature, followed by washing and analysis by flow cytometry. Data was analyzed by FlowJo (Tree Star Inc.). CytoFlex flow cytometry (Beckman Coulter) was used for the analysis.

Chen Q., Chia A., Hang S.K., Lim A., Koh W.K., Peng Y., Gao F., Chen J., Ho Z., Wai L.E., Kunasegaran K., Tan A.T., Le Bert N., Loh C.Y., Goh Y.S., Renia L., Dong T., Vathsala A, & Bertoletti A. (2023). Engineering immunosuppressive drug-resistant armored (IDRA) SARS-CoV-2 T cells for cell therapy. Cellular & molecular immunology, 20(11).

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