Time course of chemotaxis and chemokinesis of neutrophils following stimulation with IL-8 or FMLP

Abstract

Polymorphonuclear cells (PMNs) attend to inflammatory sites by chemotactic movement, caused by chemoattractants (CAs) like n-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and interleukin-8 (IL-8). However, distinct but applicable assays for investigations of PMNs' migration limit in vitro examination. We integrated CD15-bead-based isolation of PMNs with analysing their chemotaxis in a novel 3D-µ-Slide migration chamber. The PMNs were exposed to different concentrations of FMLP and IL-8 (1, 10 and 100 nM) and observed for 180 min in cell-physiological environment conditions. Moving PMNs' percentage (median and interquartile range) decreased from 62% (27%) to 36% (31%) without CA, from 88% (30%) to 22% (26%) for 1 nM IL-8, from 70% (22%) to 28% (13%) for 100 nM IL-8, from 30% (23%) to 18% (46%) for 1 nM FMLP and from 76% (20%) to 28% (13%) for 100 nM FMLP. Centres of cell movement turned towards the CAs (negative values) within a single 30-min observation period: 5.37 µm (16.82 µm) without CA, −181.37 µm (132.18 µm) with 10 nM and −239.34 µm (152.19 µm) with 100 nM IL-8; −116.2 µm (69.07 µm) with 10 nM and −71.59 µm (98.58 µm) with 100 nM FMLP. FMLP and IL-8 ensure chemotaxis without increase of chemokinesis. 3D-µ-Slide chemotaxis chambers facilitate time course analyses of PMNs' migration in stable conditions over a long time with concise distinction of chemotaxis and chemokinesis.

Impact of hydroxyethyl starch and modified fluid gelatin on granulocyte phenotype and function

“…Cells were investigated by light and fluorescence microscopy in type I collagen‐filled chambers (μ‐Slide Chemotaxis) according to the manufacturer's protocol. Briefly, these slides consisted of three channels that were filled with cells embedded in collagen gel (1.5 mg/mL PureCol, Advanced BioMatrix Inc.).…”

“…The software performs cell migration analysis by recognizing and tracking single cells semi‐automatically over a defined time span. We divided the whole observation period into bundles of approximately 0.6 hours . An average of 200 to 400 cells were tracked during that time.…”

Polymorphonuclear Cell Chemotaxis and Suicidal NETosis: Simultaneous Observation Using fMLP, PMA, H7, and Live Cell Imaging

“…To observe PMN functions in vitro, several chemotactic substances are used, such as pathogen-derived N-formyl-methionine-leucyl-phenylalanine (fMLP) or host-derived IL-8 [ 12 , 13 ]. fMLP, for instance, modulates PMN adhesion [ 14 ], microtubule polymerization [ 15 ], and activation of protein kinase C (PKC) isoforms [ 16 ] via its receptor-associated tyrosine kinases [ 17 ].…”

“…Each of the methods stated here has its advantages and disadvantages, meaning it is up to the researchers to decide which method is the most suitable for their investigation. Live cell imaging [ 12 , 22 ] uses viable neutrophils with the possibility of cell observation over any length of time and using any desired stimulus. With further development, this method could become the new gold standard, combining its advantages with solutions to the disadvantages of former methods.…”

Granulozytentransfusion: Update 2020

“…Eine Zellzählung sollte durchgeführt werden, um eine Mindestdosierung (1 × 10 10 bei Erwachsenen und 0,5 × 10 9 /kg Körpergewicht bei Neugeborenen) nicht zu unterschreiten. Eine tatsächliche Funktionsprüfung [39,42] der Granulozyten vor Infusion ist bisher nicht vorgesehen, sondern die kli- nische Wirkung wird erhofft und beobachtet, nachdem die GKs mit 30 Gy bestrahlt wurden [1].…”