Spreading of Neutrophils: From Activation to Migration

Abstract

Neutrophils rely on rapid changes in morphology to ward off invaders. Time-resolved dynamics of spreading human neutrophils after activation by the chemoattractant fMLF (formyl methionyl leucyl phenylalanine) was observed by RICM (reflection interference contrast microscopy). An image-processing algorithm was developed to identify the changes in the overall cell shape and the zones of close contact with the substrate. We show that in the case of neutrophils, cell spreading immediately after exposure of fMLF is anisotropic and directional. The dependence of spreading area, A, of the cell as a function of time, t, shows several distinct regimes, each of which can be fitted as power laws (A ~ t(b)). The different spreading regimes correspond to distinct values of the exponent b and are related to the adhesion state of the cell. Treatment with cytochalasin-B eliminated the anisotropy in the spreading.

Quantitative Reflection Interference Contrast Microscopy (RICM) in Soft Matter and Cell Adhesion

“…For certain cell types, for instance neutrophils, there may be considerable surface roughness, for example due to the presence of microvilli that scatter, rather than reflect, light. [30] Another consideration that is important while interpreting RIC images from cells is the possibility of a second reflection from the upper membrane of the cell [3,5] (see, for example, the lamellipodia in Figure 2 D). A particularly striking example is that of a red blood cell for which, after adhesion, strong fringes can be seen due to reflection from the upper membrane of the cell even though the lower membrane is tightly bound.…”

“…Thus, a combination of a variance filter to identify the non-adhering parts and an intensity threshold to identify the adhered areas has to be used. [30] The intensity threshold can be easily identified by constructing a histogram of the entire RIC micrograph. The peak of the histogram of course corresponds to the grey background intensity.…”

“…In case a time-dependent process such as cell spreading is observed, the thresholds determined for one snapshot can be consistently used to evaluate the entire RICM sequence. [30] It is clear from the above discussion that the main error in height determination in cells comes from the lack of an independent measurement of the refractive index which may, in addition, vary with time, for example with the movement of intracellular organelles. However, if we are interested in relatively fast processes that induce limited intensity variations, such as membrane fluctuation, it may be safe to assume that the re- Figure 9.…”

Occlusive thrombi arise in mammals but not birds in response to arterial injury: evolutionary insight into human cardiovascular disease

“…on June 7, 2019. by guest www.bloodjournal.org From adherent cell surface area was measured over the course of 10 minutes by the use of RICM. 30 Platelets exhibited a 3-fold increase in surface area within 2 minutes of contact with collagen and a 5-fold increase at 10 minutes ( Figure 3A and supplemental Video 3). Thrombocytes covered an area 30% greater than that of platelets at 10 minutes ( Figure 3B, supplemental Video 3), but the increase was only 2-fold at 2 minutes and 3.5-fold at 10 minutes ( Figure 3A; P Ͻ .0001 vs platelets).…”

Interações moleculares na adesão celular em suportes sólidos e o efeito de fotossensibilizadores porfirínicos

“…Apesar de alguns estudos não deixarem explícito por quanto tempo após o plaqueamento as células foram visualizadas, a maioria concentra-se na análise da adesão em um intervalo de até 2h30 após o plaqueamento [40,46,70,71,74,76,77,[81][82][83][84][85]87,92].…”