5’-bromo-2’-deoxyuridine (BrdU) detection of proliferation was performed as previously described (6 (link)). Briefly, mice were administered 0.8 mg of BrdU by i.p. injection, and maintained on BrdU in drinking water (0.8 mg/mL, plus 10% dextrose), ad libitum, from days 4-8 post-infection. Peritoneal cavity wash cells were stained for surface markers, and fixed using a BD Cytofix/Cytoperm solution, for 30 minutes at 4°C. Prior to pelleting the cells, BD Perm/Wash solution was added. Cells were then resuspended in 10% dimethyl sulfoxide (DMSO) in BD Perm/Wash solution and incubated for 10 minutes at 4°C. Cells were then fixed again with BD Cytofix/Cytoperm solution for 5 minutes at 4°C. DNase I in 1x Phosphate Buffered Saline (PBS) was then incubated on the cells for 1 hour at 37°C. The cells were then stained with FITC-conjugated anti-BrdU (Bu20a) diluted in BD Perm/Wash solution for 30 minutes at room temperature. Cells were then washed and analyzed as below.
Perm wash solution
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The Perm/Wash solution is a laboratory reagent used for the preparation of samples in various analytical procedures. It is a multi-purpose solution designed to facilitate the processing and analysis of samples in a laboratory setting. The core function of this product is to provide a consistent and reliable solution for sample preparation, without making claims about its intended use or application.
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Cytofix cytoperm solution, by BD (31 mentions)
Golgiplug, by BD (13 mentions)
Cytofix cytoperm, by BD (13 mentions)
Facscanto 2, by BD (11 mentions)
Cytofix cytoperm buffer, by BD (10 mentions)
Ionomycin, by Merck Group (9 mentions)
Golgistop, by BD (9 mentions)
Cytofix cytoperm kit, by BD (8 mentions)
Phorbol myristate acetate (pma), by Merck Group (8 mentions)
Facsdiva software, by BD (8 mentions)
Facscanto 2 flow cytometer, by BD (7 mentions)
Facscalibur, by BD (6 mentions)
Brefeldin a, by BD (6 mentions)
Lsrii flow cytometer, by BD (5 mentions)
Brefeldin a, by Merck Group (4 mentions)
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Lsrfortessa, by BD (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Monensin, by BD (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
123 protocols using perm wash solution
1
BrdU Proliferation Assay Protocol
2
Intracellular TNF-α Expression Analysis
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Intracellular TNF-α expression was analyzed using a Fixation and Permeabilization Solution Kit with BD GolgiPlugTM (BD Bioscience). Briefly, THP1 monocytes (1 × 105 cells/ml) or macrophage-like cells (about 2.0 × 105 cells/ml) were exposed to X-rays. At 24 h after X-irradiation, LPS (1 μg/ml) or FSL-1 (50 ng/ml) was added to cultures, and the cells were cultured for an additional 8 h. GolgiPlug, which inhibits protein transporters, was added to the culture medium after 2 h of an 8-h culture. Cells were harvested and fixed using Cytofix/CytopermTM solution (BD Bioscience) for 15 min on ice. After washing twice in Perm/WashTM solution (BD Bioscience), cells were suspended in Perm/WashTM solution and were stained with anti-human TNF-α-FITC mAbs or isotype control at room temperature in the dark. After 30 min, the cells were washed with Perm/WashTM solution and were analyzed using a flow cytometer.
3
Flow Cytometry Analysis of Treated Cells
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After CADA treatment, cells were collected, fixed and permeabilized with fixation/permeabilization solution (BD biosciences, Allschwil, Switzerland) and subsequently washed with perm/wash solution (BD biosciences, Allschwil, Switzerland). Next, cells were stained with antibodies and incubated at 4 °C for 45 min. Samples were then washed with perm/wash solution (BD biosciences) and fixed in PBS containing 1% formaldehyde before acquisition on BD FACS Celesta flow cytometer (Beckton Dickinson) with BD FACSDiva 8.0.1 software (BD biosciences, Allschwil, Switzerland). All data were analyzed in FlowJo X v10 (BD biosciences, Allschwil, Switzerland).
4
Multiparameter Flow Cytometry Staining
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All cells were first stained for viability using Fixable Viability Dye eFluor™ 780 (1:1000) (eBioscience). For cell surface staining, cells were incubated in FACS buffer (PBS containing 0.5% bovine serum albumin [BSA; Sigma‐Aldrich] and 0.1% NaN3) containing antibodies for 10 min at 4°C. Then, cells were fixed with 2% paraformaldehyde (PFA; Electron Microscopy Sciences) for 5 min at 4°C. For intracellular staining, the fixation step was followed by a permeabilization step in Perm/Wash solution (BD Biosciences) for 5 min at 4°C, and an intracellular staining step with antibodies diluted in Perm/Wash solution for 10 min at 4°C. The antibody clones and manufacturers used are listed in Table S1. Single‐cell measurements were performed on a FACS Canto flow cytometer (BD Biosciences) and FlowJo V10 software (TreeStar) was used to analyze the data. For each flow cytometry experiment, viable single cells were gated, after which Jurkat cells were selected on CD45 expression, and PBLs were selected on CD8 or CD4 expression. For transduction experiments, the Jurkat cells were gated on FLAG, and PBLs on Ly6G or CD90.2 (or both) and then on CD4. Exceptionally, the Ly6G+ Lck‐KD, and Ly6G+CD90.2+ Lck/Fyn‐KD PBLs were not gated on CD4, but the total T cells were assessed.
5
Th1 and Th17 Cell Differentiation
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CD4+ T cells were stimulated with plate-bound anti-CD3 (2 µg/ml) and anti-CD28 (2 µg/ml) in Th0 (neutral), Th1 (10 ng/ml IL-12 and 10 µg/ml anti–IFN-γ), or Th17 (20 ng/ml IL-6, 5 ng/ml TGFβ, 10 µg/ml anti–IFN-γ, and 10 µg/ml anti–IL-4) conditions for 3 d. Skewed cells were placed in 96-well plates and restimulated with PMA (10 ng/ml) and ionomycin (1 µg/ml) in the presence of monensin (3 µM) for 4 h at 37°C. Cells were then washed in FACS buffer (1% BSA plus 0.1% sodium azide) and fixed and permeabilized with Cytofix/Cytoperm solution for 25 min on ice, followed by washing in Perm/Wash solution (BD). Cells were stained for 30 min at room temperature with mAbs against IL-17A and IFN-γ, washed two times in Perm/Wash solution, and once with FACS buffer, and analyzed by flow cytometry (FACSCalibur using CellQuest Pro 5.2.1 software [BD]). Data were analyzed with FlowJo 9.2 software (Tree Star).
6
Mouse Stomach Cell Immunophenotyping
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The mouse stomach cells were blocked with 2 μL/106 cells of Fc Block (BD) in 100 μL fluorescence-activated cell sorter (FACS) buffer (0.5% FBS, 1 mmol/L EDTA, 0.05% NaN3 in PBS) for 10 minutes at 4°C, followed by washing with FACS buffer to remove Fc Block residue. Cell surface staining was performed in FACS buffer containing antibody cocktails (PerCP [Peridinin chlorophyll] /Cy [Cyanine] 5.5-CD45, APC [Allophycocyanin]/Cy7-CD11b, and APC-F4/80; PerCP/Cy5.5-CD45, APC/Cy7-CD11b, APC-F4/80, Pacific Blue–MHCII, and PE/Cy7-CD93; PerCP/Cy5.5-F4/80, APC/Cy7-CD11b, Pacific Blue–MHCII, PE/Cy7-CD93, APC-CD64, APC-CD21/35, APC-CD204, APC-CD14, and APC-VSIG4) on ice for 1 hour. After washing with FACS buffer 3 times, the cells were subjected to flow cytometry with a BD LSR Fortessa cytometer and data were analyzed with FlowJo software.
For intracellular staining, the Fc-blocked cell surface was stained with antibody cocktails on ice for 1 hour. After washing with FACS buffer, the cells were fixed using Cytofix/Cytoperm solution (BD) for 20 minutes on ice, followed by washing with Perm/Wash solution (BD). Intracellular staining was performed using PE-Il1β (BioLegend) for 1 hour on ice. The cells were washed again with Perm/Wash solution twice and analyzed by flow cytometry using a BD LSR Fortessa cytometer.
For intracellular staining, the Fc-blocked cell surface was stained with antibody cocktails on ice for 1 hour. After washing with FACS buffer, the cells were fixed using Cytofix/Cytoperm solution (BD) for 20 minutes on ice, followed by washing with Perm/Wash solution (BD). Intracellular staining was performed using PE-Il1β (BioLegend) for 1 hour on ice. The cells were washed again with Perm/Wash solution twice and analyzed by flow cytometry using a BD LSR Fortessa cytometer.
7
Multi-parameter immune cell profiling
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Cells were stained for viability with Viability Dye-eFluor506 (eBiosciences). For human's surface markers, cells were stained in FACS solution with the following antibodies: BDCA4-PB (clone 12C2, Biolegend), and HLA-DR-APC Vio770 (clone REA805, Miltenyi Biotech).
Cells were fixed with fixation/permeabilization solution (BD Biosciences). For intracellular staining cell were permeabilized in Perm / wash solution (BD Biosciences) and stained with: IFN-a-PE Vio615 (clone REA1013, Miltenyi Biotec), TNF-a-APC (clone Mab11, Biolegend), and IP-10-PE (clone J034D6, Biolegend). Mouse cells were stained with the following antibodies: PDCA1-PE-Cyanine7 (clone eBio927, eBiosciences) and B220-APC-Cyanine7 (clone RA3-6B2, Invitrogen). For intracellular staining, cell were permeabilized in Perm/wash solution (BD Biosciences) and stained with : anti-IFNa-APC (clone RMMA-1, Invitrogen), anti-TNFa-PE (clone MP6-XT22, BD Biosciences), and anti-IL-12p40-PE-Cy5.5 (clone C17.8, Invitrogen). Samples were acquired on LSR II or Fortessa (BD Biosciences). The gating strategy is displayed as Supporting Information Fig. S4 and Fig. S5.
Cells were fixed with fixation/permeabilization solution (BD Biosciences). For intracellular staining cell were permeabilized in Perm / wash solution (BD Biosciences) and stained with: IFN-a-PE Vio615 (clone REA1013, Miltenyi Biotec), TNF-a-APC (clone Mab11, Biolegend), and IP-10-PE (clone J034D6, Biolegend). Mouse cells were stained with the following antibodies: PDCA1-PE-Cyanine7 (clone eBio927, eBiosciences) and B220-APC-Cyanine7 (clone RA3-6B2, Invitrogen). For intracellular staining, cell were permeabilized in Perm/wash solution (BD Biosciences) and stained with : anti-IFNa-APC (clone RMMA-1, Invitrogen), anti-TNFa-PE (clone MP6-XT22, BD Biosciences), and anti-IL-12p40-PE-Cy5.5 (clone C17.8, Invitrogen). Samples were acquired on LSR II or Fortessa (BD Biosciences). The gating strategy is displayed as Supporting Information Fig. S4 and Fig. S5.
8
Characterization of WG-59 Cells by Flow Cytometry
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The WG-59 cell characterization was performed by flow cytometry at the 20th passage. In brief, 100 µL of 1 × 106 cells were incubated for 20 min at 4 °C in BD Cytofix/Cytoperm (BD Bioscience, Heidelberg, Germany) in order to fix and the cells and permeabilize the cell membrane. Cells were then washed with 10% BD Wash/Perm Solution (BD Bioscience) and incubated with antibodies against α-actin (R&D Systems, Minneapolis, MN, USA), myosin heavy chain (R&D Systems, Minneapolis, MN, USA), calponin (Acris, San Diego, CA, USA) and smoothelin (Acris, San Diego, CA, USA) in a total volume of 50 µL for 30 min. Cells were washed again in 10% BD Wash/Perm Solution and were resuspended in 300–400 µL BD Staining Buffer (BD Bioscience). The measurement was performed on a benchtop analyzer LSR II (Becton Dickenson, Heidelberg, Germany). FlowJo software (Tree Star, Inc., Ashland, OR, USA) was used for data analysis. To exclude non-specific binding, mouse IgG1 and IgG2a isotype controls (BioLegend, London, UK) were used.
9
Profiling Hematopoietic Cell Differentiation
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For analysis of KD efficiency, GFP+ cells were resorted 48 h or 72 h after lentiviral transduction. GFP+ cells were then fixed with BD Cytofix/Cytoperm Buffer (Beckton Dickinson). Subsequently, intracellular PABPN1 (AF647 anti-Pabpn1 Clone EP3000Y, abcam) staining was performed using PermWash solution (Beckton Dickinson).
Cell Cycle Analysis HSC/MPP surface staining (LSK, CD150, CD48, CD34) was performed on BM cells, Scr control/KD cells and CB-839-treated HSPCs. Cells were fixed with BD Cytofix/Cytoperm Buffer (Beckton Dickinson). Subsequently, intracellular Ki-67 (BD Biosciences) staining was performed using PermWash solution (Beckton Dickinson). Prior to flow cytometry analysis, cells were stained with Hoechst 33342 (Invitrogen) or DAPI (ThermoFisher).
In Vitro Differentiation Assay 48 h after lentiviral transduction, 1,000 GFP+ cells were resorted per condition. Scr control and Pabpn1 KD cells were cultured in 96-well ultra-low attachment plates in Complete Stem Cell Medium (specified elsewhere). 7 days later, cells were stained (anti-CD4 (clone GK1.5)-PE-Cy5, anti-CD8a (53-6.7)-PE-Cy5, anti-CD11b (M1/70)-PE-Cy7, anti-B220 (RA3-6B2)-PE-Cy5 and anti-TER119 (Ter-119)-PE-Cy5; anti-CD117/c-Kit (2B8)-BV711; anti-Ly6a/Sca-1 (D7)-APC-Cy7; anti-CD34 (RAM34)-AF700; anti-CD48 (HM48-1)-PE; anti-CD16/32 (93)-APC) and flow cytometry-based analysis was performed.
Cell Cycle Analysis HSC/MPP surface staining (LSK, CD150, CD48, CD34) was performed on BM cells, Scr control/KD cells and CB-839-treated HSPCs. Cells were fixed with BD Cytofix/Cytoperm Buffer (Beckton Dickinson). Subsequently, intracellular Ki-67 (BD Biosciences) staining was performed using PermWash solution (Beckton Dickinson). Prior to flow cytometry analysis, cells were stained with Hoechst 33342 (Invitrogen) or DAPI (ThermoFisher).
In Vitro Differentiation Assay 48 h after lentiviral transduction, 1,000 GFP+ cells were resorted per condition. Scr control and Pabpn1 KD cells were cultured in 96-well ultra-low attachment plates in Complete Stem Cell Medium (specified elsewhere). 7 days later, cells were stained (anti-CD4 (clone GK1.5)-PE-Cy5, anti-CD8a (53-6.7)-PE-Cy5, anti-CD11b (M1/70)-PE-Cy7, anti-B220 (RA3-6B2)-PE-Cy5 and anti-TER119 (Ter-119)-PE-Cy5; anti-CD117/c-Kit (2B8)-BV711; anti-Ly6a/Sca-1 (D7)-APC-Cy7; anti-CD34 (RAM34)-AF700; anti-CD48 (HM48-1)-PE; anti-CD16/32 (93)-APC) and flow cytometry-based analysis was performed.
10
Cell Cycle Analysis of Murine Leukemia Cells
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The cell cycle status of primary murine leukaemia cells was analysed using total BM or spleen cells, collected from mice with AML, sacrificed on day 40 after transplantation. The cell cycle status of different cell lines was analysed by using 5 × 105 cells, which had been serum-starved for 16 h. Cells were fixed and permeabilized with BD Cytofix/CytopermTM, washed with Perm/WashTM solution (Cat. no.: 554722, 554723; BD Biosciences, San José, CA) and stained overnight at 4 °C with an antibody to Ki-67 (BioLegend, San Diego, CA, USA). On the following day, DAPI (0.2 μg/ml) (Merck, Darmstadt, Germany) was added to each sample, and after 30 min of incubation the cells were analysed by flow cytometry (BD Fortessa, Heidelberg, Germany).
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