Jul 07, 2020

Public workspacePMN- 03 Culture of Human PMN - Migration

DOI
dx.doi.org/10.17504/protocols.io.bhrmj546
  • 1Center for Research in Medical Pharmacology, University of Insubria (Varese, Italy)
Mattia Di Rocco
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DOI: dx.doi.org/10.17504/protocols.io.bhrmj546
Protocol Citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano LM Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PMN- 03 Culture of Human PMN - Migration. protocols.io https://dx.doi.org/10.17504/protocols.io.bhrmj546
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 22, 2020
Last Modified: July 07, 2020
Protocol Integer ID: 38413
Abstract
Published work using this protocol:

- A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions.
Alex Mabou Tagne, Franca Marino, Massimiliano Legnaro, Alessandra Luini, Barbara Pacchetti, Marco Cosentino. Int J Mol Sci. 2019 Apr 13;20(8):1833. doi: 10.3390/ijms20081833.

- β 2 -Adrenoceptors Inhibit Neutrophil Extracellular Traps in Human Polymorphonuclear Leukocytes.
Franca Marino, Angela Scanzano, Laura Pulze, Monica Pinoli, Emanuela Rasini, Alessandra Luini, Raffaella Bombelli, Massimiliano Legnaro, Magda de Eguileor, Marco Cosentino . J Leukoc Biol. 2018 Sep;104(3):603-614. doi: 10.1002/JLB.3A1017-398RR. Epub 2018 Apr 18.

- Adrenergic Modulation of Migration, CD11b and CD18 Expression, ROS and interleukin-8 Production by Human Polymorphonuclear Leukocytes.
Angela Scanzano, Laura Schembri, Emanuela Rasini, Alessandra Luini, Jessica Dallatorre, Massimiliano Legnaro, Raffaella Bombelli, Terenzio Congiu, Marco Cosentino, Franca Marino. Inflamm Res. 2015 Feb;64(2):127-35. doi: 10.1007/s00011-014-0791-8. Epub 2015 Jan 6.
Materials
MATERIALS
ReagentRPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
ReagentN-FORMYL-MET-LEU-PHE (fMLP)Sigma AldrichCatalog #F3506
ReagentPropanololSigma AldrichCatalog #P0884
ReagentXyleneSigma-aldrichCatalog #247642
ReagentHematoxylinSigma – AldrichCatalog #MHS16
ReagentIL-8Sigma-aldrichCatalog #I1645
STEP MATERIALS
ReagentRPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
ReagentIL-8Sigma-aldrichCatalog #I1645
ReagentN-FORMYL-MET-LEU-PHE (fMLP)Sigma AldrichCatalog #F3506
ReagentPropanololSigma AldrichCatalog #P0884
ReagentHematoxylinSigma – AldrichCatalog #MHS16
ReagentXyleneSigma-aldrichCatalog #247642
Dubnoff bath


Protocol materials
ReagentRPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
ReagentN-FORMYL-MET-LEU-PHE (fMLP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #F3506
ReagentPropanololMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0884
ReagentHematoxylinMerck MilliporeSigma (Sigma-Aldrich)Catalog #MHS16
ReagentRPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
ReagentN-FORMYL-MET-LEU-PHE (fMLP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #F3506
ReagentHematoxylinMerck MilliporeSigma (Sigma-Aldrich)Catalog #MHS16
ReagentXyleneMerck MilliporeSigma (Sigma-Aldrich)Catalog #247642
ReagentXyleneMerck MilliporeSigma (Sigma-Aldrich)Catalog #247642
ReagentIL-8Merck MilliporeSigma (Sigma-Aldrich)Catalog #I1645
ReagentIL-8Merck MilliporeSigma (Sigma-Aldrich)Catalog #I1645
ReagentPropanololMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0884
ReagentIL-8Merck MilliporeSigma (Sigma-Aldrich)Catalog #I1645
ReagentN-FORMYL-MET-LEU-PHE (fMLP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #F3506
ReagentRPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
ReagentPropanololMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0884
ReagentHematoxylinMerck MilliporeSigma (Sigma-Aldrich)Catalog #MHS16
ReagentXyleneMerck MilliporeSigma (Sigma-Aldrich)Catalog #247642
Separation of PMN (at least 7 million PMN).
Preparation of 6 Boyden chambers:
Cut the bottom part of the vial and apply the filter (with holes of 3-5 μm in diameter) with glue / mastic (fig. 1). With paper scotch, name all Boyden chambers (fig. 2).

figure 1
figure 2

Preparation of the solutions for:
  • IL-8 10 ng/ml (2 μL stock in 198 μL di RPMI) chemotactic agent,
  • fMLP 0.1 μM (10 μL stock in 990 μL di PBS/BSA) chemotactic agent,
  • other activating stimulus according specific experimental plan,
  • drug/substances to test at different concentrations: one chamber for each concentration.

ReagentIL-8Merck MilliporeSigma (Sigma-Aldrich)Catalog #I1645

ReagentN-FORMYL-MET-LEU-PHE (fMLP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #F3506


Fill the bottom of each Boyden chamber with Amount1 mL of RPMI.

ReagentRPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml

Remove 10 μL of RPMI from each Boyden chamber and add Amount10 µL of IL-8 or fMLP treatment (except for control).

Resuspend 7x106 PMN in Amount700 µL of RPMI.

Immerse the filters in the chambers.
Put Amount100 µL of cell suspension in 4 vials and treat cells with Amount10 µL of testing drug.

Place the cells on the filters (100 μL of cell suspension on each filter) using the P100 taking care to direct the tip towards the center of the filter without touching it (if the filter does not get wet at all, gently slam the chamber).
Cap and put Boyden's chambers in the Dubnoff bath for Duration01:30:00 .

Under the chemical hood, prepare the following material:
  • 4 rows of mini becker (6 becker per row).
In each row of becker arrange, in the following order, equal quantities (2 ml in total) of: water, propanolol, xylene, propanolol + xylene (fig. 3)
  • 1 row of lids for 3-6 drops of hematoxylin to cover the same area of the filter (6 lids) and 5 plastic pasteur (each suitably marked with the name of the substance that will collect: water, propanolol, xylene, hematoxylin, immersion oil).(fig.4)

Figure 3
Figure 4

Immerse the filters related to eppendorf in:
  • PROPANOLOL for Duration00:04:00
  • WATER for Duration00:04:00
  • HEMATOXYLIN for Duration00:06:00
  • WATER for Duration00:06:00
(in the meantime throw the hematoxylin into the liquid discharge, wrap its containers in the paper and throw it all in special waste)
  • PROPANOLOL for Duration00:06:00
  • PROPANOLOL + XYLENE forDuration00:06:00
(meanwhile throw away water and propanolol in the liquid discharge and immerse the beakers to be washed in a bowl with water, alcohol and bleach taking care to turn them over so they are hygienized every side)
XYLENE for Duration00:06:00
ReagentPropanololMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0884

ReagentXyleneMerck MilliporeSigma (Sigma-Aldrich)Catalog #247642


ReagentHematoxylinMerck MilliporeSigma (Sigma-Aldrich)Catalog #MHS16

Throw away the used pasteur, wrapping it first in the paper.
Set up the slides:
nominate them,
arrange the filter (taking care to maintain the correct orientation),
arrange the cover glass (by tapping laterally with the tweezers to make sure it is well stuck),
add on top a drop of immersion oil.




Microscopy: make sure that the lens comes into contact with the immersion oil.
Critical
Once the filter with the colored cells in Haematoxylin is in focus, using only the micrometric lens bring into focus the layer of neutrophils above and report the value by saying "UP"; then you go down into the depth of the filter (rotating the micrometric lens) and bring into focus the migrated neutrophil layer: mark the visual limit value of the individual migrated cells saying "DOWN".
Imaging
The delta between the "UP" and "DOWN" values (express as μm) represents the distance in micrometers traveled by the migrating cells.
For each slide make 10 counts of the distance traveled by migrated neutrophils (selected in different optical fields): each count must be done by focusing on a different migrated neutrophil.
Results will be expressed or as absolute migration toward the filter or as ratio vs activating stimulus (sample migration/stimulus migration)