Visualization of Extracellular Traps in Macrophages from Human Bronchoalveolar Lavage Fluids

Published: October 31, 2023

Abstract

Source: Sharma, R. et al., Visualizing Macrophage Extracellular Traps Using Confocal Microscopy. J. Vis. Exp. (2017)

This video demonstrates the immunolabeling of macrophage extracellular traps, METs, in bronchoalveolar macrophages from bronchoalveolar lavage fluid, and their visualization using a confocal microscope.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Bronchoalveolar Lavage (BAL) Macrophages Use BAL to obtain lung macrophages in (1) human subjects by bronchoscopy, and (2) mice by using intra-tracheal aspiration. NOTE: Acquisition of the macrophages generally causes some activation of these cells. Macrophages are obtained from patients who require a bron…

Divulgazioni

The authors have nothing to disclose.

Materials

Human samples: Primary antibodies
Rabbit anti-human H3Cit (Citrulline R26) Abcam AB5103 10 ug/ml
Mouse anti-human MMP9 Novus Biological AB119906 1:200 ascites, no concentration given
Rabbit anti-human PADI2/PAD2 Abcam AB16478 7 ug/ml
Mouse anti-human PADI4/PAD4 Abcam AB128086 10.1 ug/ml
Secondary antibodies
Chicken anti-rabbit AF 488/ Life technologies A-21441
Chicken anti-rabbit AF 594 Life technologies A-21442
Chicken anti-goat AF 594 Life technologies a-21468
Chicken anti-mouse AF488 Life technologies A-21200
Software Programs
Imaris Bitplane
Image J NIH
To average intensity of fluorphores a standard office application like Microsoft Excel can be used
Microscopes
C1 Confocal scanning microscope Nikon
FV1200 Confocal scanning microscope Olympus
Human BAL samples from the bronchoscopy suite at Monash Medical Centre
Media
RPMI Sigma-Aldrich r8758
Fetal calf serum Sigma-Aldrich F0926
L-glutamine Sigma-Aldrich G7513
Other reagents
paraformaldehyde/periodate/lysine (PLP) fixative Sigma-Aldrich 27387
Natural formalin Sigma-Aldrich 42904
Coverslips 12 ml Sigma-Aldrich S1815
Coverslips 60 x 24 ml Sigma-Aldrich C6875

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Citazione di questo articolo
Visualization of Extracellular Traps in Macrophages from Human Bronchoalveolar Lavage Fluids. J. Vis. Exp. (Pending Publication), e21722, doi: (2023).

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