Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso
Summary
Questo manoscritto descrive un protocollo dettagliato per fenotipici e analisi quantitativa dei macrofagi residenti da reni di ratto mediante citometria di flusso. Le cellule colorate risultanti possono essere utilizzati anche per altre applicazioni, incluse separazione delle cellule, analisi di espressione genica o studi funzionali, aumentando così le informazioni ottenute nel modello sperimentale.
Abstract
There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.
Introduction
Renal disease is a global health problem, with increased prevalence, and associated with elevated morbidity and mortality1. One of the most important mechanisms involved in the progression and development of renal injury is inflammation, mainly triggered by macrophages. Macrophages play a pivotal role in many inflammatory diseases, including renal disorders2. Thus, an elevated presence of infiltrating macrophages has been reported in biopsies from patients with acute kidney injury (AKI) or chronic kidney disease (CKD)3,4. Recent studies suggest that the long-term outcome of renal disease could be controlled by macrophages5,6. In response to the local microenvironment, macrophages may differentiate into different phenotypes that play diverse biological functions7. Two well differentiated macrophage phenotypes have been established: classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2)8. M1 macrophages promote inflammation, whereas M2 macrophages have an anti-inflammatory role and are involved in tissue repair9. Therefore, a better knowledge of macrophage heterogeneity is necessary to understand their regulation and contribution to renal pathology and develop novel therapeutic approaches.
Both, murine and rats models have been widely used to understand the molecular and cellular mechanism involved in renal injury10. However, there are substantial differences in the diverse markers used to identify macrophages phenotypes between these rodents. Hence, several murine markers, such as F4/80 or Ly6C are not used in rats, thus limiting the extrapolation of findings between these species. Moreover, there is a limited number of markers describing macrophage phenotypes in rats, explaining the scarce studies analyzing macrophage heterogeneity in these animals as compared with mice. Therefore, new strategies for macrophage subset characterization are necessary to understand the role of macrophages in renal disease models in rats.
This manuscript describes a protocol for the phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. This technique can be further followed by several assays, including cell sorting and mRNA or protein expression studies to allow in-depth characterization of the role of macrophages in renal disease.
Protocol
Representative Results
Discussion
I macrofagi sono cellule eterogenee che svolgono un ruolo importante in diverse malattie infiammatorie, incluse le patologie renali. Vi è un crescente interesse nella caratterizzazione dei macrofagi sottoinsiemi nella malattia renale perché ogni sottopopolazione macrofagi contribuisce in modo diverso allo sviluppo di danno renale, come riportato nella glomerulonefrite, nefropatia diabetica e cancro del rene 14-16. Nelle fasi iniziali di danno renale acuta, una predominanza di macrofagi M1 si osserva, promuo…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from FIS/FEDER (Programa Miguel Servet: CP10/00479, PI13/00802 and PI14/00883), Spanish Society of Atherosclerosis, Spanish Society of Nephrology and Fundaciòn Renal Iñigo Alvarez de Toledo (FRIAT) to Juan Antonio Moreno. FIS/FEDER funds PI14/00386 and Instituto Reina Sofìa de Investigaciòn Nefrològica to Jesús Egido. Fundaciòn Conchita Rabago to Melania Guerrero Hue. Fundaciòn Renal Iñigo Alvarez de Toledo (FRIAT) to Alfonso Rubio Navarro.
Materials
| Laminar flow hood | Faster | Or equivalent equipment | |
| Centrifuge | Hettich | Or equivalent equipment | |
| Flow cytometer (FACSAria) | BD Biosciences | ||
| Fetal bovine serum | BioWest | S1820-500 | |
| PBS 10x | LONZA | BE17-515Q | |
| Collagenase | Sigma-Aldrich | 12/1/9001 | |
| ACK Lysing Buffer | Thermo Fisher Scientific | A10492-01 | |
| Flow cytometry strainers | BD Biosciences | 340626 | |
| Falcon cell strainers | Thermo Fisher Scientific | 352340 | |
| Flow cytometry tubes | Falcon | 352052 | 5 ml Polystyrene Round-Bottom Tube |
| Centrifuge tubes | Corning centristar | 430791 | |
| Water bath | Memmert GmbH + Co. KG | WNE 7 | 37ºC |
| Fixation/Permeabilization Solution or Permeabilization/Wash Buffer | BD Biosciences | 554714 | |
| Rompum (Xylazine) | Bayer | Or equivalent | |
| Ketalar (Ketamine) | Pfizer | Or equivalent | |
| Hanks’ balanced salt solution | Sigma-Aldrich | H8264-500ML | |
| Saline solution | Braun | 622415 | |
| Anti-CD45 (clone:OX-1) APC-Cy7 | Biolegend | 202216 | Diluted 1:100 |
| Anti-CD68 (clone: ED1) FITC | Bio-RAD | MCA341F | Diluted 35:1000 |
| anti-CD86 (clone: 24F) PE | Biolegend | 200307 | Diluted 35:1000 |
| anti-CD163 (clone: ED2) Alexa Fluor 647 | Bio-RAD | MCA342R | Diluted 4:100 |
| Live/dead stain | Molecular Probes | L34955 | Diluted 3:1000 |
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