PBMCs were isolated by density gradient centrifugation and resuspended in R10. PBMCs were rested in RPMI without glutamine and FBS (R0) for two hours. In some cases, PBMC are incubated in R10 for 1–2 days, passed through a 40µµ strainer and adjusted to 0.5 × 106/mL in PBS and rested prior to PMA stimulation. For glutamine supplementation experiments, PBMCs were rested in PBS containing 0.2mM glutamine and increasing concentrations of glutamine (0–5 mM) were added before PMA stimulation. PMA (1–20ng/mL) stimulation was performed in 1 mL PBS for 5–30 minutes in 37°C CO2 incubator. After stimulation, cells were fixed in 1.6% paraformaldehyde for 10 minutes at room temperature, spun down and resuspended in 100% cold methanol and incubated overnight at −20°C. Cells were then washed and stained with fluorochrome-conjugated phospho-antibodies: phospho-S6 (N7–548, BD Biosciences), phospho-ERK1/2 (20A, BD Biosciences), phospho-P65 (K10–895.12.50, BD Biosciences), phospho-P38 (36/p38, BD Biosciences), phospho-AKT (pS473, M89-61, BD Biosciences), IκBα (L35A5, Cell Signaling Technology), along with CD3 (HIT3a, BioLegend), CD4 (L200, BD Biosciences), CD45RO (UCHL1, BD Biosciences), CD45RA (HI100, BD Biosciences) antibodies.
Phospho p65 k10 895
Manufactured by BD
Phospho-P65 (K10–895.12.50) is a lab equipment product manufactured by BD. It is a reagent used to detect and quantify the phosphorylated form of the NF-kB subunit p65 in cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 hit3a, by BioLegend (2 mentions)
Cd4 l200, by BD (2 mentions)
Cd45ra hi100, by BD (2 mentions)
Iκbα l35a5, by Cell Signaling Technology (2 mentions)
Cd45ro uchl1, by BD (2 mentions)
Phospho s6 n7 548, by BD (2 mentions)
Phospho erk1 2 20a, by BD (2 mentions)
Phospho p38 36 p38, by BD (2 mentions)
2 protocols using phospho p65 k10 895
1
PBMC Signaling Pathway Analysis
2
PBMC Signaling Pathway Analysis
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PBMCs were isolated by density gradient centrifugation and resuspended in R10. PBMCs were rested in RPMI without glutamine and FBS (R0) for two hours. In some cases, PBMC are incubated in R10 for 1–2 days, passed through a 40µµ strainer and adjusted to 0.5 × 106/mL in PBS and rested prior to PMA stimulation. For glutamine supplementation experiments, PBMCs were rested in PBS containing 0.2mM glutamine and increasing concentrations of glutamine (0–5 mM) were added before PMA stimulation. PMA (1–20ng/mL) stimulation was performed in 1 mL PBS for 5–30 minutes in 37°C CO2 incubator. After stimulation, cells were fixed in 1.6% paraformaldehyde for 10 minutes at room temperature, spun down and resuspended in 100% cold methanol and incubated overnight at −20°C. Cells were then washed and stained with fluorochrome-conjugated phospho-antibodies: phospho-S6 (N7–548, BD Biosciences), phospho-ERK1/2 (20A, BD Biosciences), phospho-P65 (K10–895.12.50, BD Biosciences), phospho-P38 (36/p38, BD Biosciences), phospho-AKT (pS473, M89-61, BD Biosciences), IκBα (L35A5, Cell Signaling Technology), along with CD3 (HIT3a, BioLegend), CD4 (L200, BD Biosciences), CD45RO (UCHL1, BD Biosciences), CD45RA (HI100, BD Biosciences) antibodies.
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