Name: phenotypic characterization of macrophages from rat kidney by flow cytometry
Uploaded: 2016-10-18T14:00:00
Description: Watch this Scientific Journal Video about Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry at JoVE.com

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 Fundaci&#242;n Renal I&#241;igo Alvarez de Toledo (FRIAT) to Juan Antonio Moreno. FIS/FEDER funds PI14/00386 and Instituto Reina Sof&#236;a de Investigaci&#242;n Nefrol&#242;gica to Jes&#250;s Egido. Fundaci&#242;n Conchita Rabago to Melania Guerrero Hue. Fundaci&#242;n Renal I&#241;igo Alvarez de Toledo (FRIAT) to Alfonso Rubio Navarro.

이 프로토콜은 지침 유럽 의회의 63분의 2,010 / EU 및 국가 가이드 라인 2천13분의 53 다음 지역 기관 동물 케어 및 사용위원회에 의해 승인되었다. 시약 및 솔루션 1. 준비 <ol><li> 무균 조건 하에서 모든 시약 및 솔루션을 준비하고 층류 후드 사용합니다. 4 ° C에서 솔루션을 유지합니다. </li><li> 염색 버퍼를 준비 (2 % 태아 소 혈청 1 배 둘 베코의 PBS에서 (FBS)). </li><li> 식염수의 각 ml의 콜라게나 ?...

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+depletion+ameliorates+glycerol-induced+acute+kidney+injury+in+mice.">Macrophage depletion ameliorates glycerol-induced acute kidney injury in mice.</a> Nephron Exp. Nephrol. 128, 21-29 (2014).</li><li>Belliere, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+macrophage+subtypes+influence+the+progression+of+rhabdomyolysis-induced+kidney+injury.">Specific macrophage subtypes influence the progression of rhabdomyolysis-induced kidney injury.</a> J. Am. Soc. Nephrol. 26, 1363-1377 (2015).</li><li>Kinsey, G. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+and+polarization:+nomenclature+and+experimental+guidelines.">Macrophage activation and polarization: nomenclature and experimental guidelines.</a> Immunity. 41, 14-20 (2014).</li><li>Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages.">Alternative activation of macrophages.</a> Nat. Rev. Immunol. 3, 23-35 (2003).</li><li>Mosser, D. M., Edwards, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+full+spectrum+of+macrophage+activation.">Exploring the full spectrum of macrophage activation.</a> Nat. Rev. Immunol. 8, 958-969 (2008).</li><li>Ortiz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+value+of+animal+models+of+kidney+failure.">Translational value of animal models of kidney failure.</a> Eur. J. Pharmacol. 759, 205-220 (2015).</li><li>Martina, M. N., Bandapalle, S., Rabb, H., Hamad, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+double+negative+alphabeta+T+cells+from+the.">Isolation of double negative alphabeta T cells from the.</a> J. Vis. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+heme+oxygenase-adiponectin-atrial+natriuretic+peptide+axis+in+renal+function.">Role of the heme oxygenase-adiponectin-atrial natriuretic peptide axis in renal function.</a> Curr. Pharm. Des. 21, 4380-4391 (2015).</li><li>Blackbeard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+rat+spinal+microglial+response+to+peripheral+nerve+injury+as+revealed+by+immunohistochemical+image+analysis+and+flow+cytometry.">Quantification of the rat spinal microglial response to peripheral nerve injury as revealed by immunohistochemical image analysis and flow cytometry.</a> J. Neurosci. 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대 식세포는 신장 질환 등 다양한 염증 질환에 중요한 역할을 이종 세포이다. 각 모집단 식세포는 사구체 신염, 당뇨 성 신장 병증 및 신장 암 14-16에서보고 된 바와 같이, 신장 손상의 발달에 다른 방식으로 기여하기 때문에 신장 질환 식세포 서브 세트의 특성에 대한 관심이 높아지고있다. 급성 신장 손상의 초기 단계에서, M1 식세포의 우세는 관상 괴사 및 염증 촉진이 관찰된다. 그러나, ...

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.

<table border="0" cellpadding="0" cellspacing="0" width="519">
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	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Laminar flow hood</td>
			<td style="width:124px;">Faster</td>
			<td style="width:135px;"></td>
			<td style="width:143px;">Or equivalent equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Centrifuge</td>
			<td style="width:124px;">Hettich</td>
			<td style="width:135px;"></td>
			<td style="width:143px;">Or equivalent equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Flow cytometer (FACSAria)</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:135px;"></td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Fetal bovine serum</td>
			<td style="width:124px;">BioWest</td>
			<td style="width:135px;">S1820-500</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">PBS 10x</td>
			<td style="width:124px;">LONZA</td>
			<td style="width:135px;">BE17-515Q</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">Collagenase</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">12/1/9001</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">ACK Lysing Buffer</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">A10492-01</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Flow cytometry strainers</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:135px;">340626</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Falcon cell strainers</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">352340</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Flow cytometry tubes</td>
			<td style="width:124px;">Falcon</td>
			<td style="width:135px;">352052</td>
			<td style="width:143px;">5 ml Polystyrene Round-Bottom Tube</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Centrifuge tubes</td>
			<td style="width:124px;">Corning centristar</td>
			<td style="width:135px;">430791</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Water bath</td>
			<td style="width:124px;">Memmert GmbH + Co. KG</td>
			<td style="width:135px;">WNE 7</td>
			<td style="width:143px;">37&#186;C</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:119px;">Fixation/Permeabilization Solution or Permeabilization/Wash Buffer</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:135px;">554714</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Rompum (Xylazine)</td>
			<td style="width:124px;">Bayer</td>
			<td style="width:135px;"></td>
			<td style="width:143px;">Or equivalent</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Ketalar (Ketamine)</td>
			<td style="width:124px;">Pfizer</td>
			<td style="width:135px;"></td>
			<td style="width:143px;">Or equivalent</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Hanks&#8217; balanced salt solution</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">H8264-500ML</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">Saline solution</td>
			<td style="width:124px;">Braun</td>
			<td style="width:135px;">622415</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:119px;">Anti-CD45 (clone:OX-1) APC-Cy7</td>
			<td style="width:124px;">Biolegend</td>
			<td style="width:135px;">202216</td>
			<td style="width:143px;">Diluted 1:100</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">Anti-CD68 (clone: ED1) FITC</td>
			<td style="width:124px;">Bio-RAD</td>
			<td style="width:135px;">MCA341F</td>
			<td style="width:143px;">Diluted 35:1000</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:119px;">anti-CD86 (clone: 24F) PE</td>
			<td style="width:124px;">Biolegend</td>
			<td style="width:135px;">200307</td>
			<td style="width:143px;">Diluted 35:1000</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:119px;">anti-CD163 (clone: ED2) Alexa Fluor 647</td>
			<td style="width:124px;">Bio-RAD</td>
			<td style="width:135px;">MCA342R</td>
			<td style="width:143px;">Diluted 4:100</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">Live/dead stain&#160;</td>
			<td style="width:124px;">Molecular Probes</td>
			<td style="width:135px;">L34955</td>
			<td style="width:143px;">Diluted 3:1000</td>
		</tr>
	</tbody>
</table>

phenotypic characterization of macrophages from rat kidney by flow cytometry

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.

우리는 신장에서 대 식세포 침투의 증가 존재와 관련된 신 손상의 염증 실험 모델에서 대 식세포의 이질성을 분석 하였다. 이전 12보고 된이 모델에서 신장 손상, 위 스타 쥐 3 주 동안 물을 마시는 알도스테론 (1 mg의 -1 kg -1 일)을 더한 소금 (염화나트륨 1 %)의 투여에 의해 유도했다. 우리 실험 스?...

Watch this Scientific Journal Video about Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry at JoVE.com

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

이 원고는 표현형 및 유동 세포 계측법에 의해 쥐의 신장에서 거주하는 대 식세포의 정량 분석을위한 상세한 프로토콜을 설명합니다. 얻어진 염색 된 세포는 이에 실험 모델에서 얻어진 정보를 증가, 세포 선별, 유전자 발현 분석 또는 기능적 연구를 포함하는 다른 응용에 사용될 수있다.

유동 세포 계측법에 의해 쥐의 신장에서 대 식세포의 표현형 특성

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

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Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

유동 세포 계측법에 의한 중추 신경계에서 유래 된 대장 세포 및 단구 세포 유래 대 식세포의 분석

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

전체 혈액에 의해 인간 Monocyte 하위 집합의 Flow Cytometry 분석

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...