Neutrophil Based Assays
- Immune cell phagocytosis is a critical part of the immune function
- Using fluorescent plate reader techniques, we are able to measure changes in the rate of phagocytosis by a variety of immune cells
- We are able to generate both concentration dependent responses and temporal changes in the phagocytotic response
The expert team at Cellomatics can support with Neutrophil based assays within a variety of projects.
Measurement of phagocytosis by neutrophils stimulated with or without IL-8, fMLP or PMA. (A) Concentration dependent measure of the induction of phagocytosis by IL-8, fMLP or PMA in neutrophils. (B) A repeat study using the same donor to measure concentration dependent responses to IL-8, fMLP or PMA in neutrophils. Data are the mean of one donor in triplicate.
Oxidative Burst - Cytochrome C release
Neutrophils were treated with different concentrations of IL8, fMLP and PMA to investigate oxidative burst by measuring cutochrome C release. (A) Concentration dependent measure of the reduction of Cytochrome C by IL-8, fMLP and PMA in neutrophils. (B) A repeat study using the same donor to measure concentration dependent responses to IL-8, fMLP and PMA in neutrophils.
Potential Neutrophil Assays- pERK
Using HTRF experiments, Cellomatics is able to measure up regulation of ERK in a range of cell models. In (A) phosphorylation of ERK is measured in recombinant HEK cells expressing the opioid NOP receptor. Furthermore, we are able to measure antagonist efficacy by measuring the shift in the response curve using various concentrations of an antagonist. In (B) we are able to measure ERK phosphorylation in primary Human Lung Fibroblasts via the β2- adrenergic receptor interaction with salbutamol.
Potential Neutrophil Assays- β-Arrestin recruitment (TANGO assays)
Tango assays involve the development of a cell line of interest and measure the ratio of interaction between two fluorescent tagged components
Neutrophils were stained with Calcein AM and added to the top chamber of the transwell. Recombinant IL8 was then added to the bottom chamber. At the end of the assay, cells in the basal chamber were quantified. Each condition is representative of three independent biological repeats (**p<0.05, n=3±SEM).
Neutrophil CXCL5-induced chemotaxis
Measurement of primary neutrophil chemotaxis by the chemoattractant CXCL5. (A) CXCL5-induced chemotaxis as adjusted RLU (RLU-basal) values and (B) percentage increase of neutrophil chemotaxis compared to 2nM IL-8. Data are represented as n=3 ± SEM.
Neutrophil Chemotaxis Antagonist Assay
- The experiment is performed to assess the effects of antagonists with IL-8 or fMLP -induced chemotaxis assays in neutrophils, with examples to follow
- Cell type: Freshly isolated Neutrophils
- A range of concentrations of the antagonist are used to challenge the chemoattractant activity
- IL-8 + Navaraxin
- fMLP+ HCH6-1
- CXCL5 + SB225002
Navarixin inhibits IL-8 induced neutrophil chemotaxis
The inhibition of IL-8 (CXCL8) induced chemotaxis by the selective CXCR1/2 antagonist Navarixin. (A) Navarixin inhibition of IL-8 induced chemotaxis as adjusted RLU (RLU-basal) values and (B) percentage inhibition of IL-8 induced neutrophil chemotaxis by Navarixin. Data are represented as n=3 ± SEM.
HCH6-1 inhibits fMLP induced neutrophil chemotaxis
Data representing (A) HCH6-1 inhibition of fMLP induced chemotaxis as adjusted RLU (RLU-basal) values and (B) percentage inhibition of fMLP induced neutrophil chemotaxis by HCH6-1. Data are represented as n=3 ± SEM
SB225002 inhibits CXCL5 induced neutrophil chemotaxis
Data representing (A) SB225002 inhibition of CXCL5 induced chemotaxis as adjusted RLU (RLU-basal) values and (B) percentage inhibition of CXCL5 induced neutrophil chemotaxis by SB225002. Data are represented as n=3 ± SEM.
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