Cells were blocked with 1 μg anti-CD16/32 mAb (eBioscience, Hatfield, UK) in goat-serum (SIGMA-ALDRICH) and then labeled with LIVE/DEAD® Fixable Aqua Dead Cell Stain (Life Technologies), plus the following mAbs conjugated to various fluorescent labels: anti-MHC-II (IA-IE) (clone M5/114), anti-CD11b (clone M1/70), anti-F4/80 (clone BM8) (all eBioscience). For intracellular staining of phosphorylated proteins, cells were washed, fixed with 2 % paraformaldehyde (PFA), and incubated in 1x permeabilization buffer (eBioscience) with polyclonal Abs against ERK1/2, phosphorylated (P-)ERK1/2, p38, P-p38, or mAbs against RSK (clone 32D7), P-RSK (clone D5D8), CREB (clone 48H2), P-CREB (clone 87G3), P-p65 (93H1) and P-p65 (18E6) (all Cell Signaling Technology). Finally, BMMs were incubated in 1x permeabilization buffer with AlexaFluor® 488 conjugated Goat anti-Rabbit Ab (Life Technologies). All flow cytometry was acquired using the Cyan ADP analyser (DakoCytomation, Stockport, UK), or BD LSR Fortessa analyser (BD Biosciences, Oxford, UK), and data analyzed using FlowJo software v7.6.5 (Tree Star, Inc, Ashland, Oregon, USA).
P p65 18e6
Manufactured by Cell Signaling Technology
The P-p65 (18E6) product is an antibody that recognizes the phosphorylated form of the p65 subunit of the NF-kB transcription factor. It is used for the detection and quantification of activated NF-kB in cellular samples.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (2 mentions)
Anti cd16 32 mab, by Thermo Fisher Scientific (2 mentions)
Alexafluor 488 conjugated goat anti rabbit ab, by Thermo Fisher Scientific (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Lsr fortessa analyser, by BD (1 mentions)
Cyan adp analyser, by Agilent Technologies (1 mentions)
Anti cd11b clone m1 70, by Thermo Fisher Scientific (1 mentions)
Cyan adp analyzer, by Agilent Technologies (1 mentions)
Lsrfortessa analyzer, by BD (1 mentions)
Anti f4 80 clone bm8, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Merck Group (1 mentions)
2 protocols using p p65 18e6
1
Multiparameter Immunophenotyping of Macrophages
2
Multiparameter Flow Cytometry Analysis of Macrophage Signaling
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Cells were blocked with 1 μg anti-CD16/32 mAb (eBioscience, Hatfield, U.K.) in goat serum (Sigma-Aldrich) and then labeled with LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies), plus the following mAbs conjugated to various fluorescent labels: anti–MHC class II (MHC-II; IA-IE) (clone M5/114), anti-CD11b (clone M1/70), and anti-F4/80 (clone BM8) (all from eBioscience). For intracellular staining of phosphorylated proteins, cells were washed, fixed with 2% paraformaldehyde, and incubated in 1× permeabilization buffer (eBioscience) with polyclonal Abs against ERK1/2, p-ERK1/2, p38, p-p38, or mAbs against RSK (clone 32D7), p-RSK (clone D5D8), CREB (clone 48H2), p-CREB (clone 87G3), p-p65 (93H1), and p-p65 (18E6) (all from Cell Signaling Technology). Finally, BMMs were incubated in 1× permeabilization buffer with Alexa Fluor 488–conjugated goat anti-rabbit Ab (Life Technologies). All flow cytometry was acquired using the Cyan ADP analyzer (DakoCytomation, Stockport, U.K.), or BD LSR Fortessa analyzer (BD Biosciences, Oxford, U.K.) and data analyzed using FlowJo software v7.6.5 (Tree Star, Ashland, OR).
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