For surface flow cytometry and cell sorting of mouse PB, BM, and spleen, red blood cells (RBCs) were lysed and stained with monoclonal antibodies in PBS plus 1 % BSA for 1 hr on ice. For flow cytometry of erythroid lineage, BM or splenic cells were stained without RBC lysis. Intracytoplasmic staining was performed with the Fixation/Permeabilization Solution kit from BD Biosciences. Cell cycle analysis was performed with the PE Mouse Anti-Ki-67 Set (BD Biosciences) and the FIX & PERM Cell Permeabilization Kit (Invitrogen). Apoptosis analysis was performed with the PE Annexin V Apoptosis Detection Kit I (BD Biosciences). DAPI was used for both cell cycle and apoptosis analysis. See Supplemental Information for antibodies. Cell populations were analyzed using an LSR Fortessa (Becton Dickinson) and sorted with a FACSAria II instrument (Becton Dickinson). Data were analyzed with FlowJo software (Tree Star).
Pe mouse anti ki 67 set
Manufactured by BD
Sourced in United States
The PE Mouse Anti-Ki-67 Set is a lab equipment product that detects the expression of the Ki-67 protein, a cellular marker for proliferation. It provides a tool for researchers to measure cell division and growth in various biological samples.
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Lab products found in correlation
Facsaria 2 instrument, by BD (2 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Anti cd8 antibody, by Thermo Fisher Scientific (1 mentions)
Anti cd4 antibody, by BioLegend (1 mentions)
Fixable viability stain 700, by BD (1 mentions)
Sca 1 pe cy7, by Thermo Fisher Scientific (1 mentions)
Canto 1, by BD (1 mentions)
Anti cd11b pe m1 70, by BD (1 mentions)
Countess 2 fl amqaf1000, by Thermo Fisher Scientific (1 mentions)
Facsdiva 8, by BD (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
5 protocols using pe mouse anti ki 67 set
1
Mouse Blood and Tissue Cytometry
2
Multiparameter T Cell Analysis
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Fresh cell suspension from the spleen was firstly stained with Fixable Viability Stain 700 (BD), anti-CD4 antibody (Biolegend) and anti-CD8 antibody (eBioscience); then PE Mouse Anti-Ki-67 Set (BD Pharmingen™, cat# 556027) was used to detect Ki-67 level in T cells.
3
Multiplex Flow Cytometry Analysis of Fetal Liver Cells
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The following antibodies were obtained from BD Biosciences (San Diego, CA, USA): anti-CD3-APC (145-2C11), anti-cKit-Pe-Cy7 (2B8), and anti CD11b-PE (M1/70). For Lineage depletion these markers were used: CD3 (145-2C11), CD4 (L3T4), CD8 (53-6.7), CD11b (M1/70), Gr1 (RB6-8C5), B220 (Ra3-6B2), Ter119 (Ly76) and Nk1.1 (PK136) biotin and subsequently were stained with streptavidin eFluor 450 (48-4317) from eBioscience (Vienna, Austria). The following antibodies were also purchased from eBioscience: Ly5.1-PE-Cy7 (A20), Ly5.2 Alexa Fluor 780 (104), B220 PE-Cy7 (RA3-6B2), Gr1 eFluor 450 (RB6-8C5) and Sca1 PE-Cy7 (D7). Cells were stained in fluorescence activated cell sorter (FACS) buffer (PBS, 2% bovine serum albumin, 0.1% sodium azide) for 30 min at 4 °C. Ultimately, cells were washed and measured either on a Canto I, or an Aria (BD Biosciences) FACS. For FL LSK, Mac1 was precluded from the lineage gate, as FL LSK express Mac1.45 (link) For apoptosis analysis, E14 FL cells were stained with 7AAD/AnnexinV kit (BD Bioscience) in combination with LSK staining. For proliferation analysis E14 FL cells were stained with PE mouse anti-Ki67 set (BD Pharmingen, San Diego, CA, USA) in combination with LSK staining or for cell cycle analysis with an adapted protocol for combined LSK and propidium iodide staining.46 (link) Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
4
Quantifying LKS Cell Proliferation
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Total cell counts were performed using a Thermo Fisher Scientific Countess II FL (AMQAF1000) and cell counting chamber slides (C10283) following the manufacturer’s instructions on days 1, 2, 4, and 8 after expansion setup. Flow cytometry was used to back-calculate LKS cell numbers specifically and to perform cell cycle analyses by combining a PE Mouse Anti-Ki-67 Set (BD Biosciences, 556027) with our LKS staining protocol. Briefly, cells were fixed in 80% vol/vol methanol in PBS at each time point, then washed in blocking buffer at 250 g for 10 min, and resuspended in a 1:50 solution of FcR Blocking Reagent in blocking buffer for 10 min at 4 °C to prevent non-specific antibody binding. Blocked cell suspensions were co-stained for 30 min at 4 °C using fluorochrome-conjugated antibodies against Ki67, CD45, Sca1, cKit, and a Lineage Cocktail at a concentration of 0.2 μg per 106 cells. Stained cell suspensions were washed once and resuspended in blocking buffer with 1 μg ml−1 DAPI in order to fully ascertain cell cycle phase using a BD FACSAria II instrument and BD FACSDiva 8.0.1.
5
Proliferative Index Determination in AML
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