The protein aggregation assay was performed by collecting 20 × 106 whole bone marrow cells and staining them with FACS antibodies against the cell surface markers for HSCs and other haematopoietic populations as described above. After washing, cells were resuspended and fixed with Cytofix/Cytoperm Buffer (BD, 5227892) on ice for 10 min. The cells were then permeabilized with BD Cytoperm Permeabilization Buffer Plus (BD, 5227892). After washing with 1×Perm/Wash Buffer (BD, 5227892), the cells were resuspended with 1×Perm/Wash Buffer containing ProteoStat detection reagent (1/5,000; Enzo, ENZ-51023-KP050) and stained for 30 min. After washing with FACS Buffer, the cells were resuspended in FACS Buffer containing DAPI (2.5 μg ml−1). Aggregation was detected with channel 582/15(488) by flow cytometry with a BD Fortessa.
Cytoperm permeabilization buffer plus
Manufactured by BD
Sourced in United States
BD Cytoperm Permeabilization Buffer Plus is a laboratory reagent used to permeabilize cells, allowing for the detection of intracellular proteins and other molecules. The buffer solution facilitates the entry of antibodies or other detection probes into the cells, enabling the analysis of intracellular targets.
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Lab products found in correlation
Cytofix cytoperm buffer, by BD (12 mentions)
Perm wash buffer, by BD (7 mentions)
Cytofix cytoperm kit, by BD (5 mentions)
Fitc brdu flow kit, by BD (5 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Brdu flow kit, by BD (2 mentions)
Apc brdu flow kit, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Rabbit anti γh2ax, by Cell Signaling Technology (2 mentions)
Rabbit anti nox4, by Proteintech (2 mentions)
Fitc conjugated goat anti rabbit antibody, by Abcam (2 mentions)
2 7 dichlorodihydrofluorescein diacetate dcf da, by Merck Group (2 mentions)
Proteostat detection reagent, by Enzo Life Sciences (1 mentions)
Fortessa, by BD (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Fixation permeabilization buffer, by BD (1 mentions)
Bromodeoxyuridine (brdu), by BD (1 mentions)
Fc block, by BioLegend (1 mentions)
24 protocols using cytoperm permeabilization buffer plus
1
Protein Aggregation Assay for Bone Marrow Cells
2
BrdU Incorporation Assay for Colon Cancer Cells
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BrdU incorporation into DNA was detected using an FITC BrdU Flow Kit (BD Biosciences, San Jose, CA). Colon cancer cells were labeled by adding BrdU solution into culture medium to produce a final BrdU concentration of 10 μM. The labeled cells were incubated for 1 h at 37°C, collected by trypsinization, centrifuged, and fixed in BD Cytofix/Cytoperm Buffer. Cells were then permeabilized with BD Cytoperm Permeabilization Buffer Plus, and fixed again with BD Cytofix/Cytoperm Buffer. BrdU epitopes were exposed by re-suspending the tumor cells in DNase (30 μg DNase/106 cells) at 37 °C for 1 h. BrdU was stained with a fluorochrome-conjugated anti-BrdU antibody (1:50 dilution, room temperature for 20 min). Total DNA was stained with 20 μl of a 7-aminoactinomycin D [7-AAD] solution (provided with the Kit) using a 5 min incubation. Tumor cells were then resuspended in 1 ml PBS. Stained cells were subsequently analyzed using a FACS Calibur Flow Cytometer (Becton Dickinson, San Jose, CA) equipped with a 488-nm laser capable of detecting both 7-AAD and FITC.
3
BrdU Proliferation and Cell Cycle Assay
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Proliferation assay and cell cycle analysis was determined using the BrdU Flow Kits (BD Pharmingen) following manufacturer’s instructions. Add 10 μL of BrdU solution (1 mM) directly to each mL of tissue culture medium and incubate the treated cells for 45 min. Fix and permeabilize the cells with BD Cytofix/Cytoperm Buffer and BD Cytoperm Permeabilization Buffer Plus. Then treat cells with DNase to expose incorporated BrdU. Stain BrdU and intracellular antigens with fluorescent antibodies. Stain total DNA for cell cycle analysis. Cells were analyzed on a FACScan flow cytometer (BD Biosciences) and data were interpreted using the Flowjo software (Treestar).
4
Cell Cycle Analysis of Renilla and TASL KO HaCaT Cells
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Renilla KO and TASL KO HaCaT cells were plated at a density of 5 × 105 in a 6-well plate. For the cell cycle analysis, BrdU flow kits (552598, BD Bioscience, USA) were used. Renilla KO and TASL KO HaCaT cells were incubated in 10 µM BrdU in growth medium for 20 min. After incubation, cells were harvested and fixed in 1x BD Cytofix/Cytoperm Buffer. The fixed cells were washed in 1x BD Perm/Wash buffer, resuspended in BD Cytoperm Permeabilization Buffer Plus, and incubated for 10 min on ice. Subsequently, the cells were washed with 1xBD Perm/Wash buffer, resuspended in 100 µL Cytofix/Cytoperm Buffer for 5 min, washed once again with 1xBD Perm/Wash buffer, and treated with diluted DNase for 30 min. The cells were washed and incubated with anti-BrdU-FITC antibody (20 min, room temperature). Following the incubation, the cells were washed with 1 mL of 1x BD Perm/Wash buffer and resuspended in 20 µL of the 7-aminoactinomycin (7-AAD) solution. The DNA content was analyzed using a BD FACSVerse Flow Cytometer and FlowJo Software (BD Biosciences, San Jose, CA).
5
Thymocyte BrdU Incorporation Assay
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Animals were injected with 200 µg BrdU (Sigma-Aldrich) i.p. After 24 h, thymi were harvested and processed to a single-cell suspension as described. For detection of BrdU incorporation, thymocytes were fixed with BD Fixation/Permeabilization buffer (BD Biosciences) for 20 min at room temperature followed by incubation with BD Cytoperm Permeabilization Buffer Plus (BD Biosciences) for 10 min on ice. Cells were then refixed with BD Fixation/Permeabilization buffer (BD Biosciences) for 5 min at room temperature. Thymocytes were treated with 30 µg DNase I for 1.5 h at 37°C to expose BrdU epitopes. Flow cytometric staining was then performed with BrdU (Bu20a).
6
Cell Cycle Analysis of Activated T Cells
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mTh1 cells (0.5-1x106) were plated on anti-CD3/CD28 coated 48-well plates for 48h in the presence of DMSO or 2.5µM HLCL65. Cells were incubated with 10µM BrdU for the last 4 h at 37°C (BrdU APC flow kit, BD 552598). For controls, 3µM aphidocolin for G1/S arrest or 0.25µM paclitaxel for G2/M arrest were added 15 min prior to addition of BrdU. Cells were stained as described in manufacturer’s instructions. Briefly, cells were blocked 10 min at 4°C in FC block (Biolegend). Primary antibodies, CD4-e450 (48-0042-82, eBioscience clone RM4-5) and CD44-FITC (103006, Biolegend, clone IM7) were added and incubated 15 min at 4°C. Cells were washed, fixed with BD Fixation Solution for 20 min at 4°C, then washed with BD Perm Wash and stored overnight in FACS. The next day, they were washed in BD Perm Wash, incubated in BD CytoPerm Permeabilization Buffer Plus 10 min at 4°C, refixed with BD Fixation solution, 5 min at 4°C, incubated with DNase solution 1h at 37°C, washed and stained with BrdU 20 min at RT, washed and resuspended with 7-AAD to stain total DNA. Cells were then run on a FACSCanto flow cytometer (BD) and analyzed by FlowJo software.
7
BrdU Labeling and Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes
8
Cell Cycle Analysis by Dual Nucleotide Pulse
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Cell suspensions were resuspended in PBE and incubated on ice for 30 min with fluorescently labeled antibodies (Table S2 ) along with 1 mg/ml anti-CD16/32 (24G2; eBioscience).
For detection of cells in early S of the cell cycle, we performed dual nucleotide pulse and staining as previously described (Gitlin et al., 2014 (link)). Briefly, mice were injected i.v. with 1 mg of EdU (A10044; Thermo Fisher Scientific) and 1 h later with 2 mg BrdU (B5002; Sigma). 30 min after the second injection, LNs were harvested, and single-cell suspensions were prepared. After cell surface receptor staining as described above, cells were fixed and permeabilized using BD Cytofix/Cytoperm fixation and permeabilization solution and BD Cytoperm Permeabilization Buffer PLUS, respectively. EdU and BrdU incorporation into DNA was assayed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) and FITC BrdU Flow Kit (BD), respectively.
For single-cell sorting, cells were stained as above and index-sorted directly into 96-well plates containing Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol using a BD FACS Aria II. Each plate contained all conditions assayed in each replicate. Cells were washed, filtered, and resuspended in PBE before analysis or sorting on BD FACS LSR II, FACS Symphony, or FACS ARIA II cytometers. All data were analyzed using FlowJo software v.10.
For detection of cells in early S of the cell cycle, we performed dual nucleotide pulse and staining as previously described (Gitlin et al., 2014 (link)). Briefly, mice were injected i.v. with 1 mg of EdU (A10044; Thermo Fisher Scientific) and 1 h later with 2 mg BrdU (B5002; Sigma). 30 min after the second injection, LNs were harvested, and single-cell suspensions were prepared. After cell surface receptor staining as described above, cells were fixed and permeabilized using BD Cytofix/Cytoperm fixation and permeabilization solution and BD Cytoperm Permeabilization Buffer PLUS, respectively. EdU and BrdU incorporation into DNA was assayed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) and FITC BrdU Flow Kit (BD), respectively.
For single-cell sorting, cells were stained as above and index-sorted directly into 96-well plates containing Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol using a BD FACS Aria II. Each plate contained all conditions assayed in each replicate. Cells were washed, filtered, and resuspended in PBE before analysis or sorting on BD FACS LSR II, FACS Symphony, or FACS ARIA II cytometers. All data were analyzed using FlowJo software v.10.
9
BrdU Proliferation Assay for Cultured Cells
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Bromodeoxyuridine (BrdU) cell proliferation assay kit (BD Biosciences) was used to detect the proliferation of cultured cells. Cells (106) were stimulated with BrdU (3 μg/ml) for 12 h and then stained with surface antigens. Harvested cells were fixed and permeabilized with BD Cytofix/Cytoperm Buffer. Next, the cells were incubated with BD Cytoperm Permeabilization Buffer Plus. Then the cells were re-fixed and treated with DNase to expose incorporated BrdU. Finally, the cells were stained with BrdU and intracellular antigens to detect the proliferation activity.
10
Splenocyte Stimulation and BrdU Incorporation Assay
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2–5 × 106 splenocytes were stimulated with 1μg/ml sHEL or 10μg/ml anti-IgM F(ab)2 (Jackson Immunoresearch) at 37 °C for 5 min and subsequently fixed and permeabilized using the BD Cytofix/Cytoperm kit in combination with Cytofix and Perm Wash buffer (BD Biosciences; 554655, 554722, 557885) before staining in intracellular antibody staining cocktail.
To detect BrdU incorporation, surface marker stained cells were fixed using BD Cytofix/Cytoperm, treated with BD Cytoperm Permeabilization Buffer Plus (BD Biosciences; 561651) by protocol, and fixed again before treatment with 30μg DNase/106 cells for 1 h at 37 °C. Cells were further incubated with fluorescent anti-BrdU prior to acquisition.
To detect BrdU incorporation, surface marker stained cells were fixed using BD Cytofix/Cytoperm, treated with BD Cytoperm Permeabilization Buffer Plus (BD Biosciences; 561651) by protocol, and fixed again before treatment with 30μg DNase/106 cells for 1 h at 37 °C. Cells were further incubated with fluorescent anti-BrdU prior to acquisition.
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