Cytofix cytoperm plus fixation permeabilization solution kit

Manufactured by BD

Sourced in France, United States

The BD Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit is a laboratory product designed for the fixation and permeabilization of cells. It enables the intracellular staining of proteins for flow cytometric analysis.

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Cytofix/Cytoperm kit (1908 citations) Cytofix/Cytoperm solution (605 citations) Fixation/Permeabilization Solution Kit (312 citations) BD Cytofix/Cytoperm Fixation/Permeabilization Kit (302 citations) Fixation/Permeabilization Kit (198 citations) Fixation/permeabilization solution (188 citations) BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (179 citations) Cytofix/Cytoperm Plus kit (161 citations) Cytofix/Cytoperm Plus (66 citations) BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (61 citations)

8 protocols using cytofix cytoperm plus fixation permeabilization solution kit

Intracranial Glioma Model with Tim-3 Overexpression

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SB-NC or SB-Tim3 OE cells (2 × 10⁴ cells), and GL261-NC or GL261-Tim3 OE cells (2 × 10⁵ cells) were intracranially injected into 6 weeks old male C57BL/6N mice. Doxycycline (2 mg/mL) were added into drinking water to induce Tim-3 overexpression at day 7 after injection. Tumor tissues were harvested at day 14 and followed by FACS and qPCR analyses. The antibody panel and qPCR primer sequences were listed in key resources table. For FACS, single cell suspension was prepared and first stained with cell membrane fluorescein-conjugated antibodies. Before intracellular fluorescein-conjugated antibody staining, cells were treated with BD Cytofix/Cytoperm™ PlusFixation/Permeabilization Solution Kit. And BD Pharmingen™Transcription Factor Buffer Set was applied for the pretreatment before intranuclear fluorescein-conjugated antibody staining. For IFN-γ detection, cells were treated with BD GolgiPlug, Ionomycin and PMA for 4 hours. BD LSRFortessa flow cytometer was employed for data acquiring. FlowJo was used for FACS data analysis. For the detection of TAMs source and polarization status in SB mouse samples, RT-qPCR was performed to detect the marker expression of microglia, peripheral monocyte, pro-inflammatory/anti-tumorigenic macrophage and anti-inflammatory/pro-tumorigenic macrophage polarization markers.

Guo Q., Shen S., Guan G., Zhu C., Zou C., Cao J., Cheng W., Xu X., Yu J., Lin Z., Wang G., Chen L., Cheng P, & Wu A. (2022). Cancer cell intrinsic TIM-3 induces glioblastoma progression. iScience, 25(11), 105329.

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Quantification of Th17 Cells by Flow Cytometry

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After centrifuging at 1500 r/min for 5 min, cells were isolated and washed in phosphate buffered saline (PBS). After added with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 and allophycocyanin (APC)-conjugated anti-CD3 (BD PharMingen, New Jersey, America), the cells were incubated at 4 °C for 30 min. Intracellular staining was then performed with the BD Cytofix/Cytoperm Plus fixation/permeabilization solution kit following the manufacturer’s instructions (BD Biosciences) and using phycoerythrin (PE)-conjugated anti-IL-17A (BD PharMingen, New Jersey, America). Flow cytometric analysis was performed to detect Th17 cells. Results were presented as total number of IL-17A + cells among the CD3 + CD4 + .

Wang Z., Wang J., Yang P., Song X, & Li Y. (2022). Elevated Th17 cell proportion, related cytokines and mRNA expression level in patients with hypertension-mediated organ damage: a case control study. BMC Cardiovascular Disorders, 22, 257.

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Flow Cytometry Analysis of hESC-Derived Cells

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Cell aggregates derived from hESCs were allowed to settle in microcentrifuge tubes and washed with PBS. Cell aggregates were incubated with Accutase at room temperature until a single-cell suspension was obtained. Cells were washed with 1 mL ice-cold flow buffer comprised of 0.2% BSA in PBS and centrifuged at 200 g for 5 min. BD Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit was used to fix and stain cells for flow cytometry according to the manufacturer's instructions. Briefly, cell pellets were re-suspended in ice-cold BD Fixation/Permeabilization solution (300 µL per microcentrifuge tube). Cells were incubated for 20 min at 4°C. Cells were washed twice with 1 mL ice-cold 1 × BD Perm/Wash Buffer and centrifuged at 10°C and 200 × g for 5 min. Cells were re-suspended in 50 µL ice-cold 1 × BD Perm/Wash Buffer containing diluted antibodies, for each staining performed. Cells were incubated at 4°C in the dark for 1–3 hr. Cells were washed with 1.25 mL ice-cold 1X BD Wash Buffer and centrifuged at 200 × g for 5 min. Cell pellets were re-suspended in 300 µL ice-cold flow buffer and analyzed in a FACSCanto II (BD Biosciences). Antibodies used were PE-conjugated anti-PDX1 (1:10 dilution, BD Biosciences); and AlexaFluor 647-conjugated anti-NKX6.1 (1:5 dilution, BD Biosciences). Data were processed using FlowJo software v10.

Geusz R.J., Wang A., Chiou J., Lancman J.J., Wetton N., Kefalopoulou S., Wang J., Qiu Y., Yan J., Aylward A., Ren B., Dong P.D., Gaulton K.J, & Sander M. (2021). Pancreatic progenitor epigenome maps prioritize type 2 diabetes risk genes with roles in development. eLife, 10, e59067.

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Huh-7 Cell Infection Cytometry Protocol

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Twenty hours prior to infection, 1 × 10⁴ Huh-7 cells per well were seeded in 96-well microplates and maintained in DMEM supplemented with 2% FBS and 0.5 g/ml gentamycin. Huh-7 cell monolayers were infected at MOI 0.5, as mentioned above for 72 h, and processed for cytometry. Briefly, cells were treated with BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Solution Kit with BD GolgiPlug™ according to the manufacturer´s instructions and then washed and incubated with 4G2-FITC antibody (donated by the Functional Genetics of Infectious Diseases Unit, Institute Pasteur, Paris) for 30 min at 4 °C. After incubation, cells were washed once with PBS (pH 7.2), resuspended, and then analyzed using a FACScan flow cytometer (BD Accuri™ C6). Mock-infected cells were used as negative controls. At least 1000 cells were gated by light scatter. Data analysis was performed with FlowJo 10 software.

Gutiérrez-Barbosa H., Castañeda N.Y, & Castellanos J.E. (2019). Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells. The Brazilian Journal of Infectious Diseases, 24(1), 13-24.

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Multiparameter Flow Cytometry Panel

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The following directly conjugated antibodies were used to identify cellular markers: αVδ1 APC (REA173), αVδ1 FITC (TS8.2), αVd2 PE (B6), αVδ2 PerCP (B6), αTCRαβ BV421 (IP26), αTCRγδ PE (5A6.E9), αTCRγδ PE-Cy5 (5A6.E9), αCD3 APC (UCHT1), αCD3 APC-Cy7 (UCHT1), αCD3 PE-Cy7 (UCHT1), αCD3 V450 (UCHT1), αCD4 APC (RPA-T4), αCD4 BV786 (SK3), αCD8a BUV496 (RPA-T8), αCD8a BV510 (RPA-T8), αCD8a BV650 (RPA-T8), αCD45 BV711 (HI30), αCD45RA BV510 (HI100), αCD69 PE (FN50), αCD69 PE-CF594 (FN50), αCD103 BUV395 (Ber-ACT8), αCD107a BUV395 (H4A3), αCCR7 PE-Cy7 (G043H7), αNKp44 APC (p44–8), αNKp44 PE (p44–8), αNKp46 BV605 (9E2), αNKp46 PE (9E2), αNur77 PE (12.14), αMyc-Tag PE (9B11), and αHA-Tag Alexa Fluor 647 (6E2). For intracellular cytokine detection, cells were fixed/permeabilized using a BD Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit (BD Biosciences) and stained with the following directly conjugated antibodies: αIFN-γ APC (4S.B3) and αTNF-α PE-Cy7 (MAb11). Dead cells were excluded from the analysis using LIVE/DEAD Fixable Aqua or LIVE/DEAD Fixable Near-IR (Thermo Fisher Scientific). All flow cytometry data were analyzed using FlowJo software (version 10.2, Tree Star).

Mayassi T., Ladell K., Gudjonson H., McLaren J.E., Shaw D.G., Tran M.T., Rokicka J.J., Lawrence I., Grenier J.C., van Unen V., Ciszewski C., Dimaano M., Sayegh H.E., Kumar V., Wijmenga C., Green P.H., Gokhale R., Jericho H., Semrad C.E., Guandalini S., Dinner A.R., Kupfer S.S., Reid H.H., Barreiro L.B., Rossjohn J., Price D.A, & Jabri B. (2019). Chronic Inflammation Permanently Reshapes Tissue-Resident Immunity in Celiac Disease. Cell, 176(5), 967-981.e19.

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Evaluating CAR-T Cell Cytokine Secretion and Cytotoxicity

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To determine the capacity of secretion in IFN-γ and IL-2 upon target cell contact, CAR-T cells were co-cultured with the target CD123⁺CAL-1 cells at an E:T ratio of 1:1 for 15 h at 37 °C. Intracellular cytokine staining was performed with anti-human IFN-γ-BV421 and IL-2-PE using the BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Solution Kit with BD GolgiPlug™ (BD Biosciences), in accordance with the manufacturer’s information. The cytotoxicity of UCB and PB CAR-T, or C0 T cells, was evaluated after labeling by using a fixable viability Dye eFluor solution, in accordance with the manufacturer’s protocol (Invitrogen), and the cells were co-cultured with the target CD123⁺ CAL-1 cells at the indicated E:T ratio of 1:1, or 5:1, for 24 h at 37 °C for each experiment. Cytotoxicity was evaluated as previously described [35 (link)]. Targeted cell death was evaluated using 7-AAD labeling.

Caël B., Galaine J., Bardey I., Marton C., Fredon M., Biichle S., Poussard M., Godet Y., Angelot-Delettre F., Barisien C., Bésiers C., Adotevi O., Pouthier F., Garnache-Ottou F, & Bôle-Richard E. (2022). Umbilical Cord Blood as a Source of Less Differentiated T Cells to Produce CD123 CAR-T Cells. Cancers, 14(13), 3168.

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Aortic Single Cell Isolation and Flow Cytometry

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Single cell suspensions were prepared from aortas as previously described.^{3 (link), 20 (link)} Briefly, the entire aorta with surrounding perivascular fat was minced with fine scissors and digested with 1 mg/mL collagenase A, 1 mg/ml collagenase B, and 100 µg/ml DNase I in phenol-free RPMI 1640 medium with 5% FBS for 30 min at 37°C, with intermittent agitation. Fc receptors were blocked with anti-mouse CD16/CD32 for 20 min at 4°C (BD Biosciences, clone 2.4G2) prior to the staining of surface markers. The antibodies used were: PerCP-Cy5 anti-CD45; PE anti-CD34; APC anti-CD34, APC-Cy7 anti-Sca-1/Ly-6A; PE-Cy7 anti-c-kit/CD117; Amcyan anti-CD31. All aortic cells were incubated with 1.5 µl of each antibody in 100 µl of FACS buffer for 35 minutes. The cells were then washed twice with FACS buffer and immediately analyzed on a FACSCanto flow cytometer with DIVA software (Becton Dickinson). Dead cells were excluded from analysis with a fixable Violet dead cell stain. Intracellular staining was then performed with the Cytofix/Cytoperm™ Plus fixation/permeabilization solution kit (BD Biosciences) using a mouse monoclonal antibody (Abcam) conjugated to Alexa Fluor 488 or 647 (Molecular Probes). For each experiment, we performed flow minus one (FMO) controls for each fluorophore to establish gates. Data analysis was performed using FlowJo software (Tree Star, Inc.).

Wu J., Montaniel K.R., Saleh M.A., Xiao L., Chen W., Owens G.K., Humphrey J.D., Majesky M.W., Paik D.T., Hatzopoulos A.K., Madhur M.S, & Harrison D.G. (2015). The Origin of Matrix-Producing Cells that Contribute to Aortic Fibrosis in Hypertension. Hypertension, 67(2), 461-468.

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Single-cell Immune Profiling by Flow Cytometry

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Single-cell preparation of each tissue was incubated with anti-mouse CD16/32 Fc block (BioLegend, USA) and LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen, USA) on ice in the dark for 15 min. Then, the cells were washed with PBS and stained with surface staining antibodies on ice in the dark for 20 min. For intracellular staining, the cells were stimulated with 10 μg/mL lipopolysaccharide (LPS), 50 ng/mL phorbol 12-myristate 13-acetate (PMA), 500 ng/mL ionomycin (Sigma) and 2 μM monensin (eBioscience, USA) for 5 h before surface staining. Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit (BD, USA) was utilized prior to incubation with intracellular antibodies. The following antibodies purchased from Biolegend or Invitrogen were used for flow cytometry: CD11b APC or eF506, Gr1 PC5 or FITC, Ly6C PE, Ly6G PC7 or SB600, CD4 PE/Dazzle, CD8 AF700, CD19 SB780, IL-10 APC, and CD25 PC7. All data were collected on Fortessa (BD) or Cytoflex (Beckman, USA) and analyzed with FlowJo software (TreeStar, version X, USA).

Cheng X., Chi L., Lin T., Liang F., Pei Z., Sun J, & Teng W. (2023). Exogenous monocyte myeloid-derived suppressor cells ameliorate immune imbalance, neuroinflammation and cognitive impairment in 5xFAD mice infected with Porphyromonas gingivalis. Journal of Neuroinflammation, 20, 55.

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