Perm wash

Manufactured by BD

Sourced in United States, United Kingdom, Germany

The Perm/Wash is a laboratory equipment used for the processing and washing of samples. It performs functions such as mixing, agitating, and separating samples during various laboratory procedures. The core function of the Perm/Wash is to assist in the preparation and processing of samples for analysis and testing.

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Lab products found in correlation

Cytofix cytoperm, by BD (160 mentions) Golgistop, by BD (33 mentions) Golgiplug, by BD (26 mentions) Cytofix, by BD (19 mentions) Facscanto 2, by BD (17 mentions) Cytofix cytoperm solution, by BD (16 mentions) Lsrfortessa, by BD (16 mentions) Facscalibur, by BD (16 mentions) Ionomycin, by Merck Group (16 mentions) Lsr 2 flow cytometer, by BD (15 mentions) Fix perm, by BD (14 mentions) Perm wash, by Thermo Fisher Scientific (11 mentions) Perm wash buffer, by BD (10 mentions) Brefeldin a, by Merck Group (10 mentions) Lsrfortessa flow cytometer, by BD (10 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions) Lsrii flow cytometer, by BD (7 mentions) Sp5 confocal microscope, by Leica (7 mentions) Cytofix cytoperm kit, by BD (6 mentions) Fix perm solution, by BD (6 mentions)

346 protocols using perm wash

Measuring TNFα Production in PMA-Stimulated U97 Cells

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TNFα production was measured 24 h after IC addition to PMA-stimulated U97 cells. Five h prior to preparation a mixture of PMA (50 ng/mL), ionomycin (1 ug/mL), and brefeldin-a (×1000 dilution according to manufacturer protocol), also known as a PIB cocktail, was added to the cell cultures. Then, the cells were centrifuged at 300 rcf for 5 min at 4 °C, and supernatant was collected and stored at −20 °C until further use. BD Fix-Perm was added, and the cells were then incubated for 20 min on ice. An equal amount of BD Perm-Wash was added, and the cells were then centrifuged at 300 rcf for 5 min at 4 °C and the supernatant was discarded. The washing step was repeated with double the amount of BD Perm-Wash. Then, anti-TNFα antibody PE conjugate was added to the cells in BD Perm-Wash and incubated on ice for 30 min, washed out three times with BD Perm-Wash, and measured via flow cytometry. In all cases, the geometric mean of the fluorescent signal was calculated and compared.

Gyebrovszki B., Ács A., Szabó D., Auer F., Novozánszki S., Rojkovich B., Magyar A., Hudecz F., Vékey K., Drahos L, & Sármay G. (2022). The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis. International Journal of Molecular Sciences, 23(10), 5828.

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Optimized Immunophenotyping by Flow Cytometry

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For intracellular and surface marker staining, cells were dissociated using 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco, 25300054), fixed for 15 min in BD Cytofix (BD, 554714) and washed 3× in PBS with 3% w/v bovine serum albumin (BSA) (MilliporeSigma, A305). Samples were then aliquoted in a 96-well plate at 5 × 10⁵ cells per well, permeabilized in BD Perm/Wash (BD, 554723) solution for 15 min, pelleted via centrifugation at 300 g, and the supernatant was aspirated. Samples were incubated for 45 min in antibodies diluted in BD Perm/Wash at a concentration of 10⁷ cells per ml per dilution in the dark and washed 3× in BD Perm/Wash before flow cytometry experiment was performed. Colour compensation was performed on each run utilizing BD anti-mouse immunoglobulin (BD, 552843) and fluorescently conjugated antibodies. All flow cytometry measurements were performed on the BD LSRFortessa cell analyser. Flow files (.fcs) were processed in FlowJo 10.6.1 (gating strategy included in Supplementary Fig. 1). Plots were generated with Prism 8.4.0. Fluorescently conjugated antibodies utilized in flow cytometry assays are listed in Supplementary Table 1.

Skylar-Scott M.A., Huang J.Y., Lu A., Ng A.H., Duenki T., Liu S., Nam L.L., Damaraju S., Church G.M, & Lewis J.A. (2022). Orthogonally induced differentiation of stem cells for the programmatic patterning of vascularized organoids and bioprinted tissues. Nature biomedical engineering, 6(4), 449-462.

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Comprehensive Immunophenotyping of B Cell Subsets

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For analysis of TLR expression on B cell subsets, mononuclear cells were stained with CD19 (APC-Cy7), CD27 (PerCP-Cy5.5), CD38 (BV-605), IgD (BV-421) and TLR4 (FITC). For staining of intracellular TLRs, cells were subsequently fixated with 4% paraformaldehyde (Alfa Aesar; Ward Hill, MA, USA) in PBS for 30 minutes at 4°C. Fixated cells were washed in 1X BD Perm/Wash (BD Biosciences, Frederick, MD, USA) and stained with TLR7 (PE) and TLR9 (APC) in 1X Perm/Wash. Stained cells were washed with Perm/Wash and subsequently with PBS before measurement of fluorescent intensity on an LSR Fortessa (BD Biosciences; Frederick, MD, USA). For purity analysis of B cells isolated using magnetic beads, cells were stained with CD20 (PE-Cy7), CD3 (FITC), CD14 (APC-Cy7), CD27 (PerCP-Cy5.5), CD40 (APC), and IgD (BV-421) specific Abs, and subsequently resuspended in PBS with 1% paraformaldehyde before measurement of fluorescent intensity on an LSR Fortessa. Fluorescent intensities were analyzed using Flowjo software version 10 (Tree Star Inc; Ashland, OR, USA).

Pettengill M.A., van Haren S.D., Li N., Dowling D.J., Bergelson I., Jans J., Ferwerda G, & Levy O. (2016). Distinct Toll-like receptor-mediated cytokine production and immunoglobulin secretion in human newborn naïve B cells. Innate immunity, 22(6), 433-443.

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Quantifying DV2 Infection in Vero E6 Cells

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Vero E6 cells were seeded in 6-well plates for 20 h and then infected with DV2 or DV2/mCherry at a MOI of 0.1. At 1, 3 and 5 dpi, cells were collected, stained with Zombie aqua (Biolegend, #423102) for exclusion of dead cells, and fixed with 2% of paraformaldehyde. For blocking and permeabilization, cells were resuspended with Fixation/Permeabilization (BD, #554714) solution at 4 • C for 20 min. For indirect detection of DV2 E protein, cells were then centrifuged and washed twice in 1 × BD Perm/Wash (BD, #554714) buffer prior to incubation at 4 • C with 4G2 (1:500) for 1 h. After two times of resuspension with 1 × BD Perm/Wash buffer, cells were stained with anti-mouse IgG antibody conjugated with AF488 (1:500) for 30 min at 4 • C. After incubation, cells were washed three times in 1 × BD Perm/Wash™ buffer. Before flow cytometric analysis, cells were filtered through a mesh to remove cell clumps.

Li L.H., Kaptein S.J.F., Schmid M.A., Zmurko J., Leyssen P., Neyts J, & Dallmeier K. (2020). A dengue type 2 reporter virus assay amenable to high-throughput screening. Antiviral research, 183.

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Intracellular Cytokine Staining of Antigen-Specific T Cells

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Blood from immunized mice was collected (100 uL per mouse) in EDTA-containing tubes, followed by ACK lysis of red blood cells. Cells were pelleted on a 96-well plate and washed with PBS once. Trp1, Trp2, or gp100 peptides (10 ug/mL) were added to cells in complete RPMI media and incubated for 2 hours in 37 C. Brefeldin A (eBioscience) was added at the recommended dilution (1:1000), and cells were incubated for another 4 hours at 37°C, for a total of 6 hours with peptides, followed by a single PBS wash. Cells were stained with fixable live/dead aqua (Life Technologies) for 15 minutes at 4 degrees, then washed once with flow cytometry buffer (PBS 1% BSA 5 mM EDTA). Antibodies staining extracellular proteins (CD4, CD8, etc.) were added at 1:100 dilution to the cell pellets and incubated for 15 minutes at 4°C, followed by a single wash with flow cytometry buffer. Cells were fixed using BD Cytofix for 15 minutes at 4°C, and washed with BD Perm Wash. Antibodies against IFN-γ and TNF-α were added at a 1:75 dilution in BD Perm Wash for 30 minutes at 4°C, followed by BD Perm Wash twice. Cells were resuspended in flow cytometry buffer and analyzed on a BD LSR Fortessa.

Yang Y.S., Moynihan K.D., Bekdemir A., Dichwalkar T.M., Noh M.M., Watson N., Melo M., Ingram J., Suh H., Ploegh H., Stellacci F.R, & Irvine D.J. (2018). Targeting small molecule drugs to T cells with antibody-directed cell-penetrating gold nanoparticles. Biomaterials science, 7(1), 113-124.

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Single-cell characterization of pancreatic cell types

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Stage 1, 4, 5, or 6 cells were dissociated to form single cells using TrypLE (Gibco) for 5 min at 37°C. Live cell staining was performed using Zombie Violet (Biolegend) diluted in PBS for 20 min at room temperature in the dark. For GP2 (stage 4), and KIT and CXCR4 (stage 1) staining, cells were stained live using antibodies in Table S1. For NKX6-1 and PDX1 (stage 4) or NEUROD1 and CHGA (stages 4, 5, and 6), cells were fixed in Cytofix/Cytoperm (BD Bioscience) for 24 h at 4°C, washed twice with Perm/Wash (BD Bioscience), and resuspended either in unconjugated primary antibodies, listed in Table S1, and incubated overnight at 4°C, or in conjugated antibodies, incubated for 30–60 min at room temperature in the dark After washing two times with Perm/Wash, secondary antibodies were added if needed, as described in Table S1, and the cells were incubated for 20–30 min at room temperature. Cells were washed in Perm/Wash; flow cytometric analysis was performed on a BD LSR Fortessa flow cytometer and Flowjo v. 10.1 was used for data analysis.

Aghazadeh Y., Sarangi F., Poon F., Nkennor B., McGaugh E.C., Nunes S.S, & Nostro M.C. (2022). GP2-enriched pancreatic progenitors give rise to functional beta cells in vivo and eliminate the risk of teratoma formation. Stem Cell Reports, 17(4), 964-978.

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Comprehensive Immune Cell Profiling

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Cell viability was assessed by staining with Zombie Green (BioLegend) or Zombie Aqua (BioLegend), diluted 1:1000 in PBS and incubated at 4°C for 30 min. For surface staining, cells were first incubated with anti-CD16/CD32 receptor antibody (BD clone 2.4G2) diluted 1:100 in PBS, for 30 min at 4°C. Cells were then stained with fluorescently conjugated antibodies diluted 1:100 in PBS for 1 h at 4°C; cell suspensions were stained with antibodies to CD3 (BD clone 145–2C11), B220 (BD clone RA3–6B2), CD4 (BD clone RM4–5), CD8a (BD clone 53–6.7), CD44 (BD clone IM7), CD62L (BD clone MEL-14), CD11b (BD clone M1/70), CD11c (BD clone N418), MHCII I-A/E (BD clone M5/114.15.2), F4/80 (BD clone T45–2342), and SIINFEKL-H2Kb tetramer (NIH Tetramer Core).
For intracellular staining, cells were fixed with BD Perm/Fix for 20 min at 4°C, and then permeabilized with BD Perm/Wash for 10 min at4°C. Cells were stained intracellularly with fluorescently conjugated antibodies to CCL2 (BD clone 2H5) and IL-6 (BioLegend clone MP5–20F3), diluted 1:100 in BD Perm/Wash, for 1 h at 4°C.
All flow cytometry was performed on a BD LSRII flow cytometer. FlowJo (Tree Star, Inc.) software was used for all flow cytometric analysis.

Orozco S.L., Daniels B.P., Yatim N., Messmer M.N., Quarato G., Chen-Harris H., Cullen S.P., Snyder A.G., Ralli-Jain P., Frase S., Tait S.W., Green D.R., Albert M.L, & Oberst A. (2019). RIPK3 Activation Leads to Cytokine Synthesis that Continues after Loss of Cell Membrane Integrity. Cell reports, 28(9), 2275-2287.e5.

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Quantifying DNA Double-Strand Breaks

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Double stranded DNA breaks were determined at 2 and 24 h after treatments by the extent of phosphorylation of the histone variant protein, H2AX, using FITC-labeled anti-γH2AX antibodies in permeabilized cells (32 (link)). Cells were washed in PBS buffer and distributed in 96 well plates (5 × 10⁵ cells/well), washed in 200 μL FACS buffer (PBS + 1% BSA), pelleted at 2000 rpm (in a Megafuge 2.0R, Heraeus Germany centrifuge) for 2 min, and fixed in 200 μL BD Cytofix/Cytoperm solution. After 15 min incubation at 4°C, cells were washed twice in 200 μL 1× BD Perm/Wash buffer, incubated at 4°C for 15 min, pelleted and resuspended in antibody solution (50 μL anti-γH2AX antibody or isotype control diluted 1: 500 in 1× BD Perm/Wash) at 4°C for 1 h, washed twice in 1× BD Perm/Wash, and resuspended in 400 μL FACS buffer. Stained and unstained control cells were analyzed by flow cytometry.

Grasso C., Fabre M.S., Collis S.V., Castro M.L., Field C.S., Schleich N., McConnell M.J, & Herst P.M. (2014). Pharmacological Doses of Daily Ascorbate Protect Tumors from Radiation Damage after a Single Dose of Radiation in an Intracranial Mouse Glioma Model. Frontiers in Oncology, 4, 356.

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Immunostaining Fixation and Permeabilization

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Cells were fixed in 2% paraformaldehyde and permeabilized in BD Perm/Wash. Stains for antibody isotypes and allotypes were prepared in BD Perm/Wash.

Noviski M., Mueller J.L., Satterthwaite A., Garrett-Sinha L.A., Brombacher F, & Zikherman J. (2018). IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate. eLife, 7, e35074.

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Evaluating ZIKV-specific PBMC Responses

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Cryopreserved PBMCs were thawed, washed and rested overnight at 37° ± 5% CO₂. CEM‐NK^R‐expressing ZIKV NS1 or DC‐SIGN (negative control) was left in media or incubated with anti‐Zika virus NS1 antibody (B4) or plasma samples at different dilutions for 1 h at 4°. PBMCs were counted, added to the culture and incubated at 37° ± 5% CO₂ in the presence of mouse anti‐human CD107a (BD 555800) and CD107b (BD 555804). After 1h, BD GolgiPlug™ (BD 555029) and GolgiStop™ (BD 554724) were added to the wells and incubated at 37° ± 5% CO₂ for another 3 h. Cells were washed and stained with surface Abs anti‐CD3, CD16, CD56 and CD69 for 30 min. Cells were washed, fixed with BD Cytofix™ and acquired on a BD LSR™ II flow cytometer or washed and resuspended in BD Cytofix/Cytoperm™ for 20 min at 4°. The cells were then washed with BD Perm/Wash™ and stained with anti‐IFN‐γ for 30 min at 4°. The cells were washed with BD Perm/Wash™ and fixed with BD Cytofix™. Samples were acquired on a BD LSR™ II flow cytometer, and the data were analysed using the FlowJo software.

Sanchez Vargas L.A., Adam A., Masterson M., Smith M., Lyski Z.L., Dowd K.A., Pierson T.C., Messer W.B., Currier J.R, & Mathew A. (2021). Non‐structural protein 1‐specific antibodies directed against Zika virus in humans mediate antibody‐dependent cellular cytotoxicity. Immunology, 164(2), 386-397.

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