BACKGROUND
animal studies are performed in controlled environments, and provide an acceptable compromise for studying events that occur in human beings, providing clues for understanding when and where such events originally occur.
for example, sufficient technological progress has now been achieved to make it possible to see, literally, when and where an acute kidney injury will induce patchy necrosis of tubular epithelial cells  <cit> .
obtaining mechanistic insight into why and how this occurs is another similarly important challenge: new drug discovery implies the ability to distinguish between the molecular causes and the biological effects in specific cell types.
in-vitro studies clearly provide an important technical complement here  <cit> .
the biological relevance of what is observed in a culture dish is, however, very uncertain, because the study environment is even more restricted.
furthermore, the cells are completely isolated from the source organ, as well as from the whole source animal.
not to mention the fact that immortalizing a cell lineage, something routinely performed to save time and money, may profoundly alter cell programming and how it responds to injury.
ex-vivo study of the cell lysate of a whole organ subjected to some form of injury would obviously be uninformative, since it would mix many different cell types and could provide misleading observations or mask significant ones.
another approach would be to examine the population of interest after the injury has been imposed in vivo .
the limitations of this approach so far have been 1) the dissociation, using enzymes at 37°c, which can dramatically modify the cell phenotype   <cit> , and 2) the heterogeneity of the dissociated cell suspension which, as far as we know, was always cultured before being studied   <cit> .
here, we describe a fast, low-cost method that keeps the cells alive and does not necessitate costly equipment, that can be used to extract thousands of intact proximal tubular cells from one or a few fresh kidneys, making it possible to carry out ex vivo and extemporaneous quantification of multiple molecular pathways or cell-type specific selection for subsequent analysis or culture .figure  <dig>
flowchart of the method detailing the three applications of dissociated cells: direct analysis, isolation of a subpopulation of cells for subsequent analysis, or for primary cell culture.
RESULTS
instantaneous, enzyme-free preparation of a cellular suspension from whole kidneys
immediately after being harvested from adult c57bl6/j mice, each kidney was immersed in dissociating buffer, chopped and dissociated using the gentlemacs cell dissociator  for 2 minutes at 4°c, with no added enzymes.
this protocol, which takes no more than 10 minutes total, is detailed in the methods section and in table  <dig> table  <dig>
protocol for direct dissociation of a whole kidney into a cell suspension
debris exclusion prior to cell analysis
the suspension contained cells of various sizes and structures, plus many cellular fragments and aggregates that would prevent proper gating using the classical fsc/ssc  plot on a cytometer.
it should be noted that debris resulting from mechanical dissociation of the kidney can lead to significant autofluorescence; this debris must be excluded in order to obtain a reliable and accurately quantitative signal.
because debris does not contain chromatin, hoechst staining was used firstly to 1) detect dna  and to exclude non-cellular debris, and 2) among intact cells, select singlets using a peak versus area plot .
autofluorescence preponderantly emits in the green channels, which is why we used the v <dig> signal to compensate for the v <dig> signal, further excluding autofluorescent debris.
this further improved the quantification of dna cellular content, and made it possible to analyze the cell-cycle .figure  <dig>
gating strategy for flow cytometry analysis of dissociated cells.
a & f. density plot displaying side scatter versus forward scatter before  and after  compensation for autofluorescence.
b & g. representation of hoechst staining in dot plots versus side scatter before  or after  compensation for autofluorescence.
c & h representation of hoechst staining in dot plots versus hoechst green signal before  or after  compensation for autofluorescence.
the compensation breaks the correlation between ssc or hoechst’s green signal with hoechst’s blue signal.
d & i. representation of the cell cycle before  and after  compensating for autofluorescence.
e & j. the first peak corresponds to g <dig> cycle cells, whereas the second peak to the g <dig> cycle cells, with cells in phase s in between.
compared to non- compensated sample , compensation  enhances the precision of the cell cycle study.
ex-vivo analysis of proximal tubular cells in the whole kidney suspension
proximal tubular epithelial cells are highly differentiated renal cells.
prominin- <dig> is a surface glycoprotein expressed in the brush border  <cit> .
anti-prominin- <dig> antibodies are theoretically appropriate for use in that a) they are commercially available , b) they have been conjugated with a number of tags, and c) they target the extracellular region of prominin- <dig>  which makes it possible to work with live, unpermeabilized cells.
there are several isoforms of prominin- <dig>  and some anti-prominin- <dig> antibodies show clone-dependent specificities for renal epithelial cellular subtypes  <cit> .
to confirm that prominin-1+ cells detected with clone mb9-3g <dig> were indeed proximal epithelial tubular cells, we measured its coexpression with megalin, a reliable marker of proximal tubules, by means of two different techniques.
using immunofluorescence and flow cytometry, we showed that prominin- <dig> and megalin colocalize .
megalin and prominin- <dig> also colocalized after ischemia reperfusion injury .figure  <dig>
immunofluorescence analysis of control  and fibrotic kidneys  at magnification x <dig>  prominin- <dig>  staining is shown in a and b, megalin  staining is shown in c and d, and double staining is shown in e and f. expression of megalin and prominin <dig> colocalize on the brush border of renal proximal tubular epithelium in mice.
in fibrotic kidneys, tubules are interspaced with fibrosis but still express prominin- <dig> and megalin.
we studied the effect of ischemia reperfusion injury on the expression of prominin- <dig> and cd <dig>  in cells from dissociated kidneys.
in control mice, prominin- <dig> was positive in  <dig>  and  <dig> % of the cells, and cd <dig> was expressed respectively in  <dig> % and  <dig> % of these prominin-1+ cells.
at day  <dig> post ischemia reperfusion injury, prominin- <dig> was positive in  <dig>  and  <dig> % of the cells, and cd <dig> was expressed respectively in  <dig> % and  <dig> % of these prominin-1+ cells .figure  <dig>
expression of cd <dig> by prominin-1
+
cells in a suspension of dissociated cells from control and ischemia reperfusion injured kidneys.
in green, cells are stained for prominin- <dig> but not for cd <dig> .
in blue, cells from control kidneys stained for prominin- <dig> and cd <dig> .
in red, cells from ischemia-reperfusion injury kidneys stained for prominin- <dig> and cd <dig> .
isolation of proximal tubular cells from a whole kidney cell suspension
we reasoned that we could capture proximal tubular cells by using magnetic microbeads conjugated with an anti-prominin- <dig> antibody that specifically targets the proximal tubules; thus, a strong magnetic field applied to the preparation would separate the proximal tubular cells from the others.
we therefore incubated the kidney cell suspension obtained as described above with microbead-conjugated anti-prominin- <dig> antibodies.
allophycocyanin -conjugated anti-prominin- <dig> antibodies were also added to label prominin-1+ cells in order to check the quality of the magnetic separation by flow cytometry.
we chose apc since its emission wavelenghth is barely affected by autofluorescence  <cit> , and because it doesn’t overlap with hoechst fluorescence spectrum.
the prominin- <dig> negative cells were eluted, and the prominin-1+ cells were flushed at the end of the procedure after the magnetic field had been lifted.
this isolation protocol is detailed in table  <dig>  the percentage of prominin-1+ cells is assessed extemporaneously by flow cytometry .
hundreds of thousand cells can be obtained per kidney.
this yield may vary depending on experimental conditions .
assessed by flow cytometry, the purity of prominin- <dig> cells in the positive fraction was  <dig>  ±  <dig> %.
to further verify the specificity of the enrichment in proximal tubular cells, we used real-time pcr to compare the quantity of cell-type specific transcripts .
we confirmed by rt-qpcr that the enriched fraction was indeed significantly enriched in prominin- <dig> mrna compared to the eluted fraction .
cd <dig> and aqp <dig> mrnas expression was very low in the enriched and the eluted fraction .table  <dig>
protocol for isolation of proximal tubular cells
specificity of proximal tubular cells enrichment.
a) density plot of prominin- <dig> expression showing an overlay of total kidney cell suspension in red , prominin-1-depleted cells in orange , and prominin- <dig> enriched cells in blue .
b) rt-pcr quantification of prominin- <dig>  cd <dig> and aqp <dig> mrnas in the eluted and the prominin- <dig> enriched fractions.
the results are shown as relative quantifications using the geometrical mean of gene gusb and rpl <dig> as the reference.
cellular culture of a specific cell type isolated from a whole kidney cell suspension
prominin-1+ cells proved suitable for primary culture.
after a period of proliferation, cells became contiguous and re-acquired an epithelial phenotype.
using contrast-phase microscopy, we observed a monolayer of polygonal, contiguous cells .
a component of epithelial tight junctions, the transmembrane protein zona occludens  <dig>  is a marker of differentiated epithelia.
zo- <dig> was found to be expressed in prominin-1+ cells .
the cells expressed mrnas of prominin- <dig> and megalin, and ctgf, but not of cd <dig> or collagen  <dig> .
when exposed to tgf-ß, collagen  <dig> and ctgf mrnas were induced and megalin mrna was down-regulated , whereas prominin- <dig> mrna remained stable.
these phenotypic changes are typical of the effect of tgf-ß on epithelial cells, and these data suggest that prominin- <dig> expression remains stable at least in the first steps of epithelial injury .figure  <dig>
primary cultures of prominin-1+ cells.
a) contrast phase microscopy of prominin-1+ cultured cells after  <dig> days.
b) immunofluorescence for zo- <dig> in prominin-1+ cultured cells.
zo- <dig> is expressed in the prominin-1+ cultured cells, with a cytoplasmic pattern, enhanced at intercellular junctions .
rt-pcr analysis of prominin-1
+
cultured cells.
a) expression of collagen- <dig>  ctgf, megalin, cd <dig> and prominin- <dig> mrnas in a primary culture of prominin-1+ cells.
prominin- <dig>  megalin and ctgf are strongly expressed as expected in proximal tubular cells, whereas collagen  <dig> and cd <dig> are virtually absent.
b) effect of tgfß on prominin- <dig>  megalin, collagen- <dig> and ctgf mrnas expression.
tgfß upregulated ctgf and collagen- <dig> mrnas, downregulated megalin mrna, and prominin- <dig> mrna remained stable.
discussion
our method can be used to analyze and separate distinct cell types from an organ  during in vivo experiments.
it yields operator-independent, quantitative and multi-parameter data on specific cell types.
this resolves the issues of the irrelevance of cell cultures to investigate organ pathology and the cell heterogeneity characteristic of in vivo studies using whole organ lysates, which makes them unsuitable for proteomics or molecular biology.
other approaches had been used previously: laser micro-dissection on frozen or fixed tissues  <cit> ; primary culture obtained by enzymatic kidney dissociation followed by facs  <cit> ; and, more recently, translating ribosome affinity purification   <cit> .
although these techniques can be useful, they all have some major drawbacks: 1) laser microdissection can only be applied to dead cells  and cannot finely differentiate between cellular subtypes ; 2) enzymatic dissociation/facs implies a long and aggressive step of dissociation at 37°c when massive changes can occur within the cells  <cit> , biasing any subsequent analysis; in addition, cell sorters are onerous equipment which are not suitable for benchtop use; 3) trap is restricted to the study of translation within cells, and needs a genetically modified strain for each cell type of interest.
our method may a have some drawbacks.
first, cell dissociation does not allow to study the interactions between different kind of cells.
second, it is dependent on the quality of the antibody used to capture epithelial cells.
third, the yield may vary depending on experimental settings, but this should be considered as informative data rather than as a bias.
in addition to allowing flow cytometry analysis and cell-type specific isolation, our methods yields live cells that can be used for cell-specific primary cultures, which may be very useful for studying the phenotype of a specific cell-type in any animal strain .
CONCLUSIONS
overall, we believe that our method offers a robust and simple tool to isolate and further study a specific cell type from an organ downstream of in vivo experimental models.
this method allows a better understanding of cell-type specific biological processes in a solid organ, and provides easy cell-specific primary cultures for any chosen animal strain.

 animal studies are performed in controlled environments, and provide an acceptable compromise for studying events that occur in human beings, providing clues for understanding when and where such events originally occur. for example, sufficient technological progress has now been achieved to make it possible to see, literally, when and where an acute kidney injury will induce patchy necrosis of tubular epithelial cells. obtaining mechanistic insight into why and how this occurs is another similarly important challenge: new drug discovery implies the ability to distinguish between the molecular causes and the biological effects in specific cell types. in-vitro studies clearly provide an important technical complement here. the biological relevance of what is observed in a culture dish is, however, very uncertain, because the study environment is even more restricted. furthermore, the cells are completely isolated from the source organ, as well as from the whole source animal. not to mention the fact that immortalizing a cell lineage, something routinely performed to save time and money, may profoundly alter cell programming and how it responds to injury. ex-vivo study of the cell lysate of a whole organ subjected to some form of injury would obviously be uninformative, since it would mix many different cell types and could provide misleading observations or mask significant ones. another approach would be to examine the population of interest after the injury has been imposed in vivo. the limitations of this approach so far have been 1) the dissociation, using enzymes at 37°c, which can dramatically modify the cell phenotype, and 2) the heterogeneity of the dissociated cell suspension which, as far as we know, was always cultured before being studied. here, we describe a fast, low-cost method that keeps the cells alive and does not necessitate costly equipment, that can be used to extract thousands of intact proximal tubular cells from one or a few fresh kidneys, making it possible to carry out ex vivo and extemporaneous quantification of multiple molecular pathways or cell-type specific selection for subsequent analysis or culture.figure flowchart of the method detailing the three applications of dissociated cells: direct analysis, isolation of a subpopulation of cells for subsequent analysis, or for primary cell culture.  instantaneous, enzyme-free preparation of a cellular suspension from whole kidneys immediately after being harvested from adult c57bl6/j mice, each kidney was immersed in dissociating buffer, chopped and dissociated using the gentlemacs cell dissociator for 2 minutes at 4°c, with no added enzymes. this protocol, which takes no more than 10 minutes total, is detailed in the methods section and in table  table protocol for direct dissociation of a whole kidney into a cell suspension debris exclusion prior to cell analysis the suspension contained cells of various sizes and structures, plus many cellular fragments and aggregates that would prevent proper gating using the classical fsc/ssc plot on a cytometer. it should be noted that debris resulting from mechanical dissociation of the kidney can lead to significant autofluorescence; this debris must be excluded in order to obtain a reliable and accurately quantitative signal. because debris does not contain chromatin, hoechst staining was used firstly to 1) detect dna and to exclude non-cellular debris, and 2) among intact cells, select singlets using a peak versus area plot. autofluorescence preponderantly emits in the green channels, which is why we used the v signal to compensate for the v signal, further excluding autofluorescent debris. this further improved the quantification of dna cellular content, and made it possible to analyze the cell-cycle.figure gating strategy for flow cytometry analysis of dissociated cells. a & f. density plot displaying side scatter versus forward scatter before and after compensation for autofluorescence. b & g. representation of hoechst staining in dot plots versus side scatter before or after compensation for autofluorescence. c & h representation of hoechst staining in dot plots versus hoechst green signal before or after compensation for autofluorescence. the compensation breaks the correlation between ssc or hoechst’s green signal with hoechst’s blue signal. d & i. representation of the cell cycle before and after compensating for autofluorescence. e & j. the first peak corresponds to g cycle cells, whereas the second peak to the g cycle cells, with cells in phase s in between. compared to non- compensated sample, compensation enhances the precision of the cell cycle study. ex-vivo analysis of proximal tubular cells in the whole kidney suspension proximal tubular epithelial cells are highly differentiated renal cells. prominin- is a surface glycoprotein expressed in the brush border. anti-prominin- antibodies are theoretically appropriate for use in that a) they are commercially available, b) they have been conjugated with a number of tags, and c) they target the extracellular region of prominin- which makes it possible to work with live, unpermeabilized cells. there are several isoforms of prominin- and some anti-prominin- antibodies show clone-dependent specificities for renal epithelial cellular subtypes. to confirm that prominin-1+ cells detected with clone mb9-3g were indeed proximal epithelial tubular cells, we measured its coexpression with megalin, a reliable marker of proximal tubules, by means of two different techniques. using immunofluorescence and flow cytometry, we showed that prominin- and megalin colocalize. megalin and prominin- also colocalized after ischemia reperfusion injury.figure immunofluorescence analysis of control and fibrotic kidneys at magnification x prominin- staining is shown in a and b, megalin staining is shown in c and d, and double staining is shown in e and f. expression of megalin and prominin colocalize on the brush border of renal proximal tubular epithelium in mice. in fibrotic kidneys, tubules are interspaced with fibrosis but still express prominin- and megalin. we studied the effect of ischemia reperfusion injury on the expression of prominin- and cd in cells from dissociated kidneys. in control mice, prominin- was positive in and % of the cells, and cd was expressed respectively in % and % of these prominin-1+ cells. at day post ischemia reperfusion injury, prominin- was positive in and % of the cells, and cd was expressed respectively in % and % of these prominin-1+ cells.figure expression of cd by prominin-1 + cells in a suspension of dissociated cells from control and ischemia reperfusion injured kidneys. in green, cells are stained for prominin- but not for cd. in blue, cells from control kidneys stained for prominin- and cd. in red, cells from ischemia-reperfusion injury kidneys stained for prominin- and cd. isolation of proximal tubular cells from a whole kidney cell suspension we reasoned that we could capture proximal tubular cells by using magnetic microbeads conjugated with an anti-prominin- antibody that specifically targets the proximal tubules; thus, a strong magnetic field applied to the preparation would separate the proximal tubular cells from the others. we therefore incubated the kidney cell suspension obtained as described above with microbead-conjugated anti-prominin- antibodies. allophycocyanin -conjugated anti-prominin- antibodies were also added to label prominin-1+ cells in order to check the quality of the magnetic separation by flow cytometry. we chose apc since its emission wavelenghth is barely affected by autofluorescence, and because it doesn’t overlap with hoechst fluorescence spectrum. the prominin- negative cells were eluted, and the prominin-1+ cells were flushed at the end of the procedure after the magnetic field had been lifted. this isolation protocol is detailed in table  the percentage of prominin-1+ cells is assessed extemporaneously by flow cytometry. hundreds of thousand cells can be obtained per kidney. this yield may vary depending on experimental conditions. assessed by flow cytometry, the purity of prominin- cells in the positive fraction was  ±  %. to further verify the specificity of the enrichment in proximal tubular cells, we used real-time pcr to compare the quantity of cell-type specific transcripts. we confirmed by rt-qpcr that the enriched fraction was indeed significantly enriched in prominin- mrna compared to the eluted fraction. cd and aqp mrnas expression was very low in the enriched and the eluted fraction.table protocol for isolation of proximal tubular cells specificity of proximal tubular cells enrichment. a) density plot of prominin- expression showing an overlay of total kidney cell suspension in red, prominin-1-depleted cells in orange, and prominin- enriched cells in blue. b) rt-pcr quantification of prominin- cd and aqp mrnas in the eluted and the prominin- enriched fractions. the results are shown as relative quantifications using the geometrical mean of gene gusb and rpl as the reference. cellular culture of a specific cell type isolated from a whole kidney cell suspension prominin-1+ cells proved suitable for primary culture. after a period of proliferation, cells became contiguous and re-acquired an epithelial phenotype. using contrast-phase microscopy, we observed a monolayer of polygonal, contiguous cells. a component of epithelial tight junctions, the transmembrane protein zona occludens is a marker of differentiated epithelia. zo- was found to be expressed in prominin-1+ cells. the cells expressed mrnas of prominin- and megalin, and ctgf, but not of cd or collagen. when exposed to tgf-ß, collagen and ctgf mrnas were induced and megalin mrna was down-regulated, whereas prominin- mrna remained stable. these phenotypic changes are typical of the effect of tgf-ß on epithelial cells, and these data suggest that prominin- expression remains stable at least in the first steps of epithelial injury.figure primary cultures of prominin-1+ cells. a) contrast phase microscopy of prominin-1+ cultured cells after days. b) immunofluorescence for zo- in prominin-1+ cultured cells. zo- is expressed in the prominin-1+ cultured cells, with a cytoplasmic pattern, enhanced at intercellular junctions. rt-pcr analysis of prominin-1 + cultured cells. a) expression of collagen- ctgf, megalin, cd and prominin- mrnas in a primary culture of prominin-1+ cells. prominin- megalin and ctgf are strongly expressed as expected in proximal tubular cells, whereas collagen and cd are virtually absent. b) effect of tgfß on prominin- megalin, collagen- and ctgf mrnas expression. tgfß upregulated ctgf and collagen- mrnas, downregulated megalin mrna, and prominin- mrna remained stable. discussion our method can be used to analyze and separate distinct cell types from an organ during in vivo experiments. it yields operator-independent, quantitative and multi-parameter data on specific cell types. this resolves the issues of the irrelevance of cell cultures to investigate organ pathology and the cell heterogeneity characteristic of in vivo studies using whole organ lysates, which makes them unsuitable for proteomics or molecular biology. other approaches had been used previously: laser micro-dissection on frozen or fixed tissues; primary culture obtained by enzymatic kidney dissociation followed by facs; and, more recently, translating ribosome affinity purification. although these techniques can be useful, they all have some major drawbacks: 1) laser microdissection can only be applied to dead cells and cannot finely differentiate between cellular subtypes; 2) enzymatic dissociation/facs implies a long and aggressive step of dissociation at 37°c when massive changes can occur within the cells, biasing any subsequent analysis; in addition, cell sorters are onerous equipment which are not suitable for benchtop use; 3) trap is restricted to the study of translation within cells, and needs a genetically modified strain for each cell type of interest. our method may a have some drawbacks. first, cell dissociation does not allow to study the interactions between different kind of cells. second, it is dependent on the quality of the antibody used to capture epithelial cells. third, the yield may vary depending on experimental settings, but this should be considered as informative data rather than as a bias. in addition to allowing flow cytometry analysis and cell-type specific isolation, our methods yields live cells that can be used for cell-specific primary cultures, which may be very useful for studying the phenotype of a specific cell-type in any animal strain.  overall, we believe that our method offers a robust and simple tool to isolate and further study a specific cell type from an organ downstream of in vivo experimental models. this method allows a better understanding of cell-type specific biological processes in a solid organ, and provides easy cell-specific primary cultures for any chosen animal strain. 
using flow cytometry, immunofluorescence, and rt-pcr, we show that our method is suitable for studying and isolating proximal tubular cells with an anti prominin- antibody. hundreds of thousands of proximal tubular cells are then isolated by magnetic separation followed by direct analysis or primary cell culture. it is also a simple method to obtain a specific primary cell culture from any animal strain. using our method, we detect phenotypic changes in the proximal tubular cells after renal ischemia reperfusion, and we isolate the proximal tubular cells, with a purity over 80%. kidneys are subjected to mechanical dissociation followed by flow-cytometry analysis. organ physiologyexperimental modelscell phenotypeprimary cell culturesin vivoin vitroissue-copyright-statement© the author 2015 here, we describe a method for analyzing and isolating specific cell types from the kidney and studying the phenotype they have acquired in vivo. experimental models are inevitably a compromise between accurately reproducing a pathological situation and schematically simplifying it, which is intended to provide both relevance and conclusiveness.
 experimental models are inevitably a compromise between accurately reproducing a pathological situation and schematically simplifying it, which is intended to provide both relevance and conclusiveness. in-vivo models are very relevant, but multiple cell-types undergoing various changes may hinder the observation of individual molecular events.  here, we describe a method for analyzing and isolating specific cell types from the kidney and studying the phenotype they have acquired in vivo. using flow cytometry, immunofluorescence, and rt-pcr, we show that our method is suitable for studying and isolating proximal tubular cells with an anti prominin- antibody. kidneys are subjected to mechanical dissociation followed by flow-cytometry analysis. hundreds of thousands of proximal tubular cells are then isolated by magnetic separation followed by direct analysis or primary cell culture. using our method, we detect phenotypic changes in the proximal tubular cells after renal ischemia reperfusion, and we isolate the proximal tubular cells, with a purity over 80%.  this method is efficient, quick, simple, and cheap, and should be useful for studying cell-type specific parameters after in vivo experimental studies. it is also a simple method to obtain a specific primary cell culture from any animal strain. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords organ physiologyexperimental modelscell phenotypeprimary cell culturesin vivoin vitroissue-copyright-statement© the author 2015 