Flow cytometry was performed as described previously (6 (link)). Isolated cells were incubated with predetermined optimized concentrations of anti-CD3-Alexa fluor 700 (clone SP34-2, BD Pharmingen), anti-CD8-Pacific Blue or PE-CF594 (clone RPA-T8, BD Pharmingen), anti-CD20-APC-Cy7 (clone L27, BD Pharmingen), anti-CXCR5-phycoerythrin (PE) (Clone MU5UBEE, eBioscience), anti-CD200-APC (clone OX104, eBioscience), anti-CD4-PerCP (clone L200, BD Pharmingen), anti-CD95-PE-Cy5 (clone DX2, BD Pharmingen), anti-CCR7-APC-Cy7 (clone 3D12, Biolegend), anti-PD-1-PE-Cy7 (clone EH12.2H7, Biolegend), anti-ICOS-Pacific Blue (C398.4A, Biolegend) and Live/Dead dye-Alexa430 (Invitrogen). After staining the cell surface, cells were permeabilized and fixed by BD perm wash (BD Pharmingen) and washed twice. Finally, cells were incubated with anti-Ki67-FITC (B56, BD Pharmingen), washed twice and diluted in 1% PFA. Data were acquired on a LSRII flow cytometer (BD Biosciences) and the data analyzed using FlowJo software (version 9.2 Tree Star).
Anti ki67 fitc b56
Manufactured by BD
Anti-Ki67-FITC (B56) is a fluorescently-labeled monoclonal antibody that binds to the Ki-67 protein, a cellular marker for proliferation. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Perm wash, by BD (1 mentions)
Anti cd3 alexa fluor 700 clone sp34.2, by BD (1 mentions)
Anti cd4 percp clone l200, by BD (1 mentions)
Anti cd95 pe cy5 clone dx2, by BD (1 mentions)
Anti pd 1 pe cy7, by BioLegend (1 mentions)
Clone eh12.2h7, by BioLegend (1 mentions)
Anti cd20 apc cy7 clone l27, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Golgistop containing monensin, by BD (1 mentions)
Anti t bet percpcy5.5 ebio4b10, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Facsymphony a3, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Macs ls column, by Miltenyi Biotec (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Fluorophore conjugated antibodies, by BD (1 mentions)
4 protocols using anti ki67 fitc b56
1
Multiparameter Flow Cytometry Assay
2
Intracellular Cytokine Analysis of Activated T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For intracellular cytokine analysis, single cell suspensions (0.5-1 million cells per well) were stimulated in 24 well plates with complete RPMI containing 20 nM phorbol myristate acetate (PMA; Sigma-Aldrich) and 1μg/ml ionomycin (Sigma-Aldrich), at 37°C for 4 hours. After 1 hour of incubation, 1μl/ml of GolgiStop (containing monensin; BD Biosciences) was added to each well. Surface stained cells were stained for intracellular antigen following Fixation/Permeabilisation (Foxp3-staining kit, eBiosciences). Intracellular antibodies consisted of anti-Ki67-FITC (B56; BD Bioscience) or anti-Ki67-BrilliantViolet 421 (16A8; BioLegend), anti-IL-2-PE (JES6-5H4; eBioscience), anti-Foxp3-PE-Cy7 (FJK-16s; eBioscience), and anti-T-bet-PerCPCy5.5 (eBio4B10; eBioscience). For flow cytometric analysis, samples were acquired on a FACS Canto II flow cytometer (BD Biosciences), and were subsequently analysed using FlowJo software.
3
Annexin-V and Ki67 Staining Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For analysis of cell death, LP cells and IELs were washed with annexin-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and incubated with fluorochrome-conjugated annexin-V and 7-AAD Viablity Staining Solution (eBioscience) for 15 min at room temperature. For analysis of cell proliferation, cells were surface-labelled with the appropriate conjugated Abs, fixed, permeabilized using Foxp3 Staining Buffer Set (eBioscience) and subsequently incubated with anti-Ki67-FITC (B56; BD Biosciences) for 30 min. After washing, cells were analysed on a FACSAria II.
4
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were stained using fluorophore-conjugated antibodies purchased from either eBiosciences, Biolegend, BD Pharmingen, or WEHI Monoclonal Antibody Production Facility. For lineage depletion, BM cells were stained with biotinylated anti-CD2, CD3, Thy1.2, Gr1, Ter119, B220, CD19, and Mac1 and depleted using MACS LS columns (Miltenyi Biotec). To distinguish apoptotic/necrotic cells in stained samples, FITC Annexin V and propidium iodide (final concentration of 2.5 μg/ml) were added 1–10 min prior to acquiring the data. For cell cycle analysis, cells were fixed and permeabilized using Cytofix/Cytoperm (BD Pharmingen) and then stained with anti-Ki67 FITC (B56; BD) and 10 µg/ml DAPI. TCR Vß-subfamily usage was assessed using the Mouse Vß TCR Screening Panel (BD Pharmingen). Cells were analyzed on an LSR Fortessa or FACSymphony A3 (BD) and sorted on a FACS Aria II (BD). Data were analyzed using FlowJo (Treestar; version 9.9.5).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!