FACS cell cycle analysis was performed with 7-AAD and Ki-67 as previously described (Goff et al., 2013 (link)). Single cell suspensions of bone marrow cells of engrafted mice with either lentiviral backbone or miR-26a conditions were immunostained with Alexa405-conjugated anti-human CD45 (Invitrogen), Alexa647-anti-human CD38 (Ab Serotec) and biotin-anti-human CD34 (Invitrogen) plus Alexa488-strepavidin (Invitrogen) in 2% fetal bovine serum/ PBS- followed by live cell staining using the LIV&OEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen). Surface stained cells were then fixed in 70% ethanol overnight and were immunostained with PE-conjugated anti-Ki-67 (BD) in 0.15% saponin/ 2% fetal bovine serum/ PBS, washed and incubated with 7-AAD (Invitrogen, 10 μg/mL in 0.1 M sodium citrate/ 5 mM EDTA pH8.0/ 0.15 M NaCl/ 0.5% BSA/ 0.02% saponin). For HEK293T cells, cells were transduced with lentiviral backbone or miR-26a for 3 days and then stained with the LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen). Cells were then fixed in 70% ethanol for 4 hr at 4°C and immunostained with PE-conjugated anti-Ki-67 (BD) and 7-AAD as described. Stained samples were analyzed using a FACSAria and FlowJo.
Pe conjugated anti ki67
Manufactured by BD
Sourced in United States
PE-conjugated anti-Ki67 is a fluorescently-labeled antibody that binds to the Ki67 protein, a cellular marker of proliferation. This product is designed for use in flow cytometry and other immunofluorescence applications to detect and quantify proliferating cells.
Automatically generated - may contain errors
Lab products found in correlation
Pe conjugated anti ros, by Abcam (2 mentions)
Fitc conjugated anti annexin 5, by BD (2 mentions)
Flow cytometry, by Beckman Coulter (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd25, by BD (1 mentions)
Percp cyanine5.5 conjugated anti cd4, by BioLegend (1 mentions)
Version 7, by FlowJo (1 mentions)
Hoechst 33342, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Pe conjugated anti cd71, by BD (1 mentions)
Pecy5 conjugated anti cd34, by BD (1 mentions)
Apc conjugated anti cd38, by BD (1 mentions)
Apc conjugated anti tnf α, by BD (1 mentions)
Pe conjugated anti granzyme b, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Transcription factor buffer set, by BD (1 mentions)
Facsaria, by FlowJo (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Fixation and permeabilization solution kit, by BD (1 mentions)
7 protocols using pe conjugated anti ki67
1
FACS Analysis of Cell Cycle and Viability
2
Comprehensive Multiparameter Immune Cell Profiling
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Peripheral blood from 7 patients in the pilot phase II study were surface stained using APC-eFluor–conjugated anti-CD19 (Thermo Fisher Scientific, catalog no. 47-0199-42, RRID:AB_1582230), PE-Texas Red–conjugated anti-CD3 (Thermo Fisher Scientific, catalog no. MHCD0317, RRID:AB_10376002), APC-conjugated anti-CD25 (BD Pharmingen, catalog no. 555434, RRID:AB_398598), PerCP/Cyanine5.5-conjugated anti-CD4 (BioLegend, catalog no. 300530, RRID:AB_893322), BV500-conjugated anti-CD45 (BD Horizon, catalog no. 560777, RRID:AB_1937324), BV605-conjugated anti-CD56 (BD Pharmingen, catalog no. 562780, RRID:AB_2728700), and BV650-conjugated anti-CD8a (BioLegend, catalog no. 301042, RRID:AB_2563505). Cells were then treated with FoxP3 fixation/ permeabilization solution and washed with permeabilization buffer (catalog no. 00-5223-00, eBioScience) using the protocol suggested by company. After cells were stained with Pacific Blue–conjugated anti-Foxp3 (BioLegend, catalog no. 320216, RRID:AB_2104902) and PE-conjugated anti-Ki67 (BD Pharmingen, catalog no. 556027, RRID:AB_2266296). As the Ki67 isotype control, the accompanying PE-conjugated IgG1 k Isotype control (BD Pharmingen, catalog no.556027 RRID:AB_2266296) was used. Data were acquired using LSRII machine. FlowJo version 10.8.1 was used to analyze the data.
3
Cell Cycle Analysis of NVP-BGJ398 Treatment
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The drug treatment was carried out as previously described with either 100 nM of NVP-BGJ398 or 0.01% DMSO. 72 h post drug treatment, cells were trypsinized and fixed with 80% cold ethanol. After PBS wash, cells were stained for 20 min with PE-conjugated anti-Ki67 (BD Biosciences, New Jersey, United States) or the corresponding isotype control (BD Biosciences, New Jersey, United States) at recommended dilutions, followed by PBS wash and Hoechst 33342 (BD Biosciences, New Jersey, United States) staining at a concentration of 2 μg/ml for 15 minutes. Staining was measured using LSR II Flow Cytometer (BD Biosciences, New Jersey, United States). For Hoechst, 355 nm UV laser with 540/50 BP filter and for PE-conjugated Ki67, 488 nm blue laser with 585/42 BP and 550 LP filters were used. The results were analyzed with FlowJo, version 7.6.5 (FlowJo, Oregon, United States). After cell doublet discrimination, the Ki67 negative cell population was gated as G0. The isotype control was used to confirm the gating strategy with Ki67. Cell cycle analysis was done using the built in software Cell Cycle Analysis module. Statistical analysis was performed using two-tailed, unpaired Student's t-Test. p-values < 0.05 were considered significant.
4
Cell Surface and Intracellular Protein Profiling
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Extracellular staining of cells was performed using PECy5–conjugated anti-CD34, APC-conjugated anti-CD38, PE-conjugated anti-CD71 (BD Biosciences) or PE-conjugated anti-GPA (Invitrogen). For intracellular staining, cells were fixed with 4% paraformaldehyde, and washed and permeabilized with 80% ethanol before adding PE–conjugated anti-P-Smad1/5/8 (Cell Signaling) or AF647-conjugated anti-P-Stat3 (Biolegend), or PE-conjugated anti-Ki67 (BD Biosciences).
5
Multiparametric analysis of CD8+ T cells
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PBMCs, isolated from a healthy volunteer, were labeled with FITC-conjugated anti-CD8 antibodies (BD Bioscience, USA) and incubated for 30 min at 4 °C. To perform intracellular staining, the cells, which had been previously labeled with antibodies against cell surface markers, underwent fixation and permeabilization using a Transcription Factor Buffer Set (BD Bioscience, USA) for a duration of 20 min to destroy the cell membrane and nuclear membrane. Afterward, they were treated with fluorochrome-conjugated antibodies, including APC-conjugated anti-TNFα (BD Pharmingen, USA), BV421-conjugated anti-IFNγ (BD Bioscience, USA), PE-conjugated anti-Ki67 (BD Bioscience, USA), and PE-conjugated anti-Granzyme B (BD Bioscience, USA). The intracellular staining was conducted at 4 °C for a duration of 30 min. In the end, the analyzed cells were run through a FACS Calibur flow cytometer provided (Becton Dickinson, USA), and the resulting data were processed using FlowJo software.
6
Quantifying Sertoli Cell Markers
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Sertoli cells were harvested to detect Ki67, Annexin V, and ROS, and the testes from the three groups were digested to generate single cells using 0.25% trypsin-EDTA. A Fixation/Permeabilization Solution Kit (BD, USA) was applied to fix and permeabilize the above cells. PE-conjugated anti-Ki67 (BD, USA), FITC-conjugated anti-Annexin V (BD, USA), PE-conjugated anti-ROS (Abcam, USA), and their corresponding isotypes were labeled for 30 min at 4 °C. Then, flow cytometry (Beckman, USA) was employed for analysis according to the manufacturer’s instructions.
7
Flow Cytometry Analysis of Sertoli Cell Markers
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To detect ROS, Ki67, and Annexin V, Sertoli cells were collected. The mouse testes were digested with 0.25% trypsin-EDTA to produce a single-cell suspension. Furthermore, the Fixation and Permeabilization Solution Kit (BD, USA) was used to fix and permeabilize the digested cells. The cells were labeled with FITC-conjugated anti-Annexin V (BD, USA), PE-conjugated anti-Ki67 (BD, USA), and PE-conjugated anti-ROS (Abcam, USA) antibodies and their isotype controls at 4 °C for 30 min. After that, flow cytometry (Beckman, USA) was used for analysis according to the manufacturer’s instructions.
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