JP2018091790A - Method for staining cell - Google Patents
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- 238000010186 staining Methods 0.000 title claims description 20
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Abstract
Description
本発明は、試料中に含まれる細胞を染色する方法に関する。特に本発明は、試料中に含まれる細胞を膜透過させた後、染色試薬を用いて前記細胞を染色する方法に関する。 The present invention relates to a method for staining cells contained in a sample. In particular, the present invention relates to a method of staining cells using a staining reagent after allowing cells contained in a sample to permeate the membrane.
細胞内における標的タンパクの局在や発現量の確認を、当該標的タンパクを認識する標識抗体を含む染色試薬で前記細胞を染色（染色工程）して確認する場合、前記染色工程前に前記細胞が有する生体膜（細胞膜や核膜など）を透過させる工程（膜透過工程）が必要である。膜透過工程では、生体膜への浸透性を増強するために一般的に界面活性剤やアルコール等が用いられる。例えば、非特許文献１では抗Ｎａｎｏｇ抗体を含む染色試薬を用いて人工多能性幹細胞（ｉＰＳ細胞）を染色する前に、界面活性剤であるサポニンを含む試薬を用いて前記細胞の膜透過処理を行っている。 When confirming the localization and expression level of a target protein in a cell by staining the cell with a staining reagent containing a labeled antibody that recognizes the target protein (staining step), the cell A step (membrane permeation step) for permeating the biological membrane (cell membrane, nuclear membrane, etc.) is required. In the membrane permeation step, a surfactant, alcohol or the like is generally used in order to enhance the permeability to the biological membrane. For example, in Non-Patent Document 1, before staining artificial pluripotent stem cells (iPS cells) with a staining reagent containing an anti-Nanog antibody, membrane permeabilization of the cells using a reagent containing a saponin as a surfactant is performed. It is carried out.
しかしながら、膜透過工程で生体膜の透過処理が不十分だと、後続の染色工程で細胞内に染色試薬が十分に拡散せず、染色が不均一になり、偽陰性が増えるという問題があった。特にｉＰＳ細胞から目的細胞に分化誘導させる工程では、ｉＰＳ細胞が造腫瘍能を有するため、分化誘導工程後に含まれるｉＰＳ細胞を極めて高感度に検出する（すなわち偽陰性を極力減らす）必要があり、より効果的な生体膜の透過処理法が求められている。 However, if the permeabilization treatment of the biological membrane is insufficient in the membrane permeation process, there is a problem that the staining reagent does not sufficiently diffuse into the cells in the subsequent staining process, the staining becomes uneven, and false negatives increase. . In particular, in the process of inducing differentiation from iPS cells to target cells, iPS cells have tumorigenic potential, and therefore it is necessary to detect iPS cells contained after the differentiation induction process with extremely high sensitivity (that is, to reduce false negatives as much as possible). There is a need for a more effective biomembrane permeabilization method.
本発明の課題は、細胞が有する生体膜の透過効率を改善し、前記細胞の染色像を改善する方法を提供することにある。 The subject of this invention is providing the method of improving the permeation | transmission efficiency of the biological membrane which a cell has, and improving the dyeing | staining image of the said cell.
上記課題を解決するために、本発明者らは鋭意検討を重ねた結果、本発明に到達した。 In order to solve the above-mentioned problems, the present inventors have intensively studied to arrive at the present invention.
すなわち、本発明の第一の態様は、以下の（１）から（４）に示す工程を含む、細胞の染色方法である。
（１）固定試薬を用いて細胞を固定する工程
（２）固定した前記細胞をアルコールを含む試薬で処理することで、前記細胞が有する生体膜の脂質成分を可溶化し透過させる工程
（３）固定した前記細胞を界面活性剤を含む試薬で処理することで、前記細胞が有する生体膜の脂質成分を可溶化し透過させる工程
（４）抗Ｎａｎｏｇ抗体を含む染色試薬を用いて膜透過した前記細胞を染色する工程
That is, the first aspect of the present invention is a cell staining method including the following steps (1) to (4).
(1) A step of fixing cells using a fixing reagent (2) A step of solubilizing and permeating lipid components of a biological membrane of the cells by treating the fixed cells with a reagent containing alcohol (3) The step of solubilizing and permeating the lipid component of the biological membrane of the cell by treating the fixed cell with a reagent containing a surfactant (4) The membrane permeated with a staining reagent containing an anti-Nanog antibody Staining cells
また、本発明の第二の態様は、固定試薬がホルムアルデヒドまたはパラホルムアルデヒドを含む、前記第一の態様に記載の染色方法である。 The second aspect of the present invention is the staining method according to the first aspect, wherein the fixing reagent contains formaldehyde or paraformaldehyde.
また、本発明の第三の態様は、アルコールがメタノールである、前記第一または第二の態様に記載の染色方法である。 Moreover, the 3rd aspect of this invention is the dyeing | staining method as described in said 1st or 2nd aspect whose alcohol is methanol.
また、本発明の第四の態様は、界面活性剤がサポニンである、前記第一から第三の態様のいずれかに記載の染色方法である。 A fourth aspect of the present invention is the staining method according to any one of the first to third aspects, wherein the surfactant is saponin.
また、本発明の第五の態様は、細胞が多能性幹細胞である、前記第一から第四の態様のいずれかに記載の染色方法である。 A fifth aspect of the present invention is the staining method according to any one of the first to fourth aspects, wherein the cell is a pluripotent stem cell.
さらに、本発明の第六の態様は、以下の（１）から（４）に示す試薬を少なくとも含むキットである。
（１）ホルムアルデヒドまたはパラホルムアルデヒドを含む固定試薬
（２）メタノールを含む生体膜透過試薬
（３）サポニンを含む生体膜透過試薬
（４）抗Ｎａｎｏｇ抗体を含む染色試薬
Furthermore, a sixth aspect of the present invention is a kit including at least the reagents shown in the following (1) to (4).
(1) Fixing reagent containing formaldehyde or paraformaldehyde (2) Biomembrane permeation reagent containing methanol (3) Biomembrane permeation reagent containing saponin (4) Staining reagent containing anti-Nanog antibody
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明において細胞とは、Ｎａｎｏｇを発現している細胞であればよく、例としてｉＰＳ細胞などの多能性幹細胞、胚性幹（ＥＳ）細胞、胚性がん（ＥＣ）細胞があげられるが、中でも多能性幹細胞が好ましい。また、細胞の状態・形状についても限定はなく、培養器材に接着している細胞や酵素処理等により分散・懸濁された細胞であってもよく、スフェロイド等の細胞塊や組織などの形状であってもよい。 In the present invention, the cell may be any cell expressing Nanog, and examples thereof include pluripotent stem cells such as iPS cells, embryonic stem (ES) cells, and embryonic cancer (EC) cells. Of these, pluripotent stem cells are preferred. In addition, there is no limitation on the state / shape of the cell, and it may be a cell adhered to a culture device or a cell dispersed / suspended by an enzyme treatment or the like, and may be in the shape of a cell mass or tissue such as a spheroid. There may be.
本発明の染色方法は、以下の（１）から（４）に示す工程を含むことを特徴としている。
（１）固定試薬を用いて細胞を固定する工程
（２）固定した前記細胞をアルコールを含む試薬で処理することで、前記細胞が有する生体膜の脂質成分を可溶化し透過させる工程
（３）固定した前記細胞を界面活性剤を含む試薬で処理することで、前記細胞が有する生体膜の脂質成分を可溶化し透過させる工程
（４）抗Ｎａｎｏｇ抗体を含む染色試薬を用いて膜透過した前記細胞を染色する工程
前記（１）の固定工程で用いる固定試薬は、ホルムアルデヒド、パラホルムアルデヒド、グルタルアルデヒドなど、当業者間で一般的に広く使用される組織高分子を架橋する試薬の中から適宜選択すればよく、好ましい固定試薬の態様として、ホルムアルデヒドまたはパラホルムアルデヒドを含む試薬があげられる。なお、前記固定試薬には前記（２）の工程で用いる試薬の主成分であるアルコールを含んではならない。
The staining method of the present invention is characterized by including the steps shown in the following (1) to (4).
(1) A step of fixing cells using a fixing reagent (2) A step of solubilizing and permeating lipid components of a biological membrane of the cells by treating the fixed cells with a reagent containing alcohol (3) The step of solubilizing and permeating the lipid component of the biological membrane of the cell by treating the fixed cell with a reagent containing a surfactant (4) The membrane permeated with a staining reagent containing an anti-Nanog antibody Step for staining cells The fixing reagent used in the fixing step (1) is appropriately selected from among reagents commonly used for cross-linking tissue polymers such as formaldehyde, paraformaldehyde, and glutaraldehyde. A preferable example of the fixing reagent is a reagent containing formaldehyde or paraformaldehyde. The fixing reagent should not contain alcohol as the main component of the reagent used in the step (2).
前記（２）および（３）の膜透過工程は、前記（１）の固定工程の後、実施する。前記（２）の膜透過工程において、アルコールは水溶性であれば特に限定はなく、メタノール、エタノール、１−プロパノール、２−プロパノールなどがあげられる、中でもエタノールやメタノールが好ましく、メタノールが特に好ましい。また、前記（２）の処理条件も当業者間で一般的に行われる条件であれば良い。例えば、−２０℃から５℃の範囲で最低５分間の処理条件が例として挙げられる。前記（３）の膜透過工程において、界面活性剤は生体膜の脂質成分を可溶化できるものであれば特に限定はなく、一例として、Ｔｒｉｔｏｎ Ｘ−１００やＴｗｅｅｎ ２０（いずれも商品名）など略無差別に脂質分子を可溶化する界面活性剤や、サポニンやジギトニンなどコレステロールを特異的に可溶化する界面活性剤が挙げられるが、中でもコレステロールを特異的に可溶化する界面活性剤が好ましく、サポニンが特に好ましい。前記（３）の条件は業者間で一般的に行われる条件であれば良く、１５℃から２５℃の範囲で１５分間から１２０分間の処理条件や１℃から１０℃の範囲で１０時間から１８時間の処理条件が例として挙げられる。 The membrane permeation steps (2) and (3) are performed after the fixing step (1). In the membrane permeation step (2), the alcohol is not particularly limited as long as it is water-soluble, and examples thereof include methanol, ethanol, 1-propanol, 2-propanol, etc. Among them, ethanol and methanol are preferable, and methanol is particularly preferable. Further, the processing condition (2) may be a condition generally performed by those skilled in the art. For example, processing conditions of a minimum of 5 minutes in the range of −20 ° C. to 5 ° C. are exemplified. In the membrane permeation step (3), the surfactant is not particularly limited as long as it can solubilize the lipid component of the biological membrane. For example, Triton X-100, Tween 20 (both are trade names), etc. Surfactants that solubilize lipid molecules indiscriminately and surfactants that specifically solubilize cholesterol, such as saponin and digitonin, can be mentioned, among which surfactants that specifically solubilize cholesterol are preferred, and saponins Is particularly preferred. The condition (3) may be a condition generally performed among contractors. The treatment condition is 15 to 120 ° C. for 15 to 120 minutes, or 10 to 18 ° C. for 1 to 10 ° C. An example is time processing conditions.
前記（４）の染色工程では、標的タンパク質であるＮａｎｏｇを特異的に標識可能な色素（化学発光色素や蛍光色素など）を添加して染色する方法も考えられるが、汎用性の点からＮａｎｏｇに対する標識抗体を用いる方法が一般的である。前記標識抗体中の標識物質は、抗原抗体反応を利用した測定の分野で通常用いられる物質の中から適宜選択すればよく、一例として、フルオレセイン等の蛍光物質や、アルカリホスファターゼ等の酵素、放射性物質があげられる。前記標識抗体における抗体と標識物質との結合態様に限定はなく、前記抗体が標識物質と化学結合などにより直接結合された態様であってもよく、標識物質が結合された前記抗体に対する抗体（標識二次抗体）によって間接的に結合された態様であってもよい。 In the staining step (4), a method of adding a dye (such as a chemiluminescent dye or a fluorescent dye) that can specifically label Nanog, which is a target protein, may be considered. A method using a labeled antibody is common. The labeling substance in the labeled antibody may be appropriately selected from substances usually used in the field of measurement using antigen-antibody reaction. Examples include fluorescent substances such as fluorescein, enzymes such as alkaline phosphatase, and radioactive substances. Can be given. The binding mode of the antibody and the labeling substance in the labeled antibody is not limited, and the antibody may be directly bound to the labeling substance by chemical bonding or the like. The antibody against the antibody to which the labeling substance is bound (label) It may be an embodiment indirectly bound by a secondary antibody).
なお、本発明の染色方法において、膜透過工程の順序に限定はなく、前記（２）の工程の後に前記（３）の工程を実施してもよいし、前記（３）の工程の後に前記（２）の工程を実施してもよい。また前記（４）の染色工程を実施する際、前記（３）の膜透過工程と同時に（すなわちＮａｎｏｇ抗体の染色試薬中に界面活性剤を含有した態様で実施してもよい。 In the staining method of the present invention, the order of the membrane permeation process is not limited, and the process (3) may be performed after the process (2), or the process after the process (3). You may implement the process of (2). Further, when carrying out the staining step (4), it may be carried out simultaneously with the membrane permeation step (3) (that is, in a mode in which a surfactant is contained in the staining reagent for Nanog antibody).
上述した染色方法を簡便に実施するために、（１）ホルムアルデヒドまたはパラホルムアルデヒドを含む固定試薬、（２）メタノールを含む生体膜透過試薬、（３）サポニンを含む生体膜透過試薬、（４）抗Ｎａｎｏｇ抗体を含む染色試薬を少なくとも含むキットを準備してもよい。 In order to easily carry out the staining method described above, (1) a fixing reagent containing formaldehyde or paraformaldehyde, (2) a biomembrane permeation reagent containing methanol, (3) a biomembrane permeation reagent containing saponin, A kit containing at least a staining reagent containing a Nanog antibody may be prepared.
本発明の染色方法および染色キットは、固定試薬で固定した細胞を、アルコールを含む試薬および界面活性剤を含む試薬で処理することで前記細胞が有する生体膜の脂質成分を可溶化し透過させた後、蛍光色素を標識した抗Ｎａｎｏｇ抗体を含む染色試薬で染色することを特徴としており、多能性幹細胞などＮａｎｏｇを発現する細胞を極めて高感度に検出する一方、偽陰性を大幅に減少できる。 In the staining method and staining kit of the present invention, cells fixed with a fixing reagent are treated with a reagent containing alcohol and a reagent containing a surfactant to solubilize and permeate lipid components of the biological membrane of the cells. Thereafter, staining is performed with a staining reagent containing an anti-Nanog antibody labeled with a fluorescent dye, and cells expressing Nanog such as pluripotent stem cells can be detected with extremely high sensitivity, while false negatives can be greatly reduced.
以下、実施例により本発明をより詳細に説明するが、当該実施例は例示のために提供されるものに過ぎず、本発明が当該実施例に示された態様に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the said Example is only provided for the illustration and this invention is not limited to the aspect shown by the said Example.
（実施例１）
膜透過工程による細胞染色の違いを以下に示す方法で評価した。
（１）ヒトｉＰＳ細胞２０１Ｂ７株（ｉＰＳアカデミアジャパンより購入）をｉＭａｔｒｉｘ（タカラバイオ社製）でコートしたシャーレに播種し、ＳｔｅｍＦｉｔＡＫ０２Ｎ培地（タカラバイオ社製）で培養した。
（２）（１）で７日間培養したｉＰＳ細胞をＰＢＳ（りん酸緩衝生理食塩水）で洗浄後、ＴｒｙｐＬＥ ｓｅｌｅｃｔ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ社製）とＶｅｒｓｅｎｅ Ｓｏｌｕｔｉｏｎ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ社製）との混合液で前記細胞を剥離した。
（３）剥離した細胞を回収後、４％（ｗ／ｖ）パラホルムアルデヒド溶液に室温で４０分間浸することで、前記細胞を固定した。
（４）固定した細胞を０．５％ＢＳＡと２ｍＭ ＥＤＴＡを含むＰＢＳ（Ｂｕｆｆｅｒ Ａ）で２回洗浄後、（ｂ）および（ｄ）の条件では９０％（ｖ／ｖ）メタノールに置換し氷上で３０分静置することで膜透過処理を行なった。
（５）（ａ）および（ｂ）の条件ではＢｕｆｆｅｒ Ａで、（ｃ）および（ｄ）の条件では５％（ｖ／ｖ）ＦＢＳ（ウシ胎仔血清）と０．７５％（ｗ／ｖ）サポニンとを含むＰＢＳ（ＢｕｆｆｅｒＢ）で、それぞれ洗浄した。
（６）（ａ）および（ｂ）の条件ではウサギ由来抗マウスＮａｎｏｇ抗体（セルシグナリングテクノロジー社製）を含むＢｕｆｆｅｒ Ａに浸し、（ｃ）および（ｄ）の条件では前記抗体を含むＢｕｆｆｅｒ Ｂに、それぞれ室温で９０分間浸すことで、未分化ｉＰＳ細胞と当該細胞が有するタンパク質（Ｎａｎｏｇ）に対する抗体との複合体を形成させた。
（７）（ａ）および（ｂ）の条件ではＢｕｆｆｅｒ Ａで、（ｃ）および（ｄ）の条件ではＢｕｆｆｅｒ Ｂで、それぞれ２回洗浄した。
（８）（ａ）および（ｂ）の条件では標識二次抗体であるＡｌｅｘａ Ｆｌｕｏｒ ４８８で標識したヤギ由来抗ウサギＩｇＧ抗体（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ社製）を含むＢｕｆｆｅｒ Ａに浸し、（ｃ）および（ｄ）の条件では前記抗体を含むＢｕｆｆｅｒ Ｂに浸し、それぞれ室温で９０分間反応させた。
（９）Ｂｕｆｆｅｒ Ａで３回洗浄後、ＦＡＣＳＡｒｉａ（ＢＤバイオサイエンス社製）にてＮａｎｏｇ陽性ｉＰＳ細胞の割合を測定した。
（１０）Ｎａｎｏｇを発現していない細胞として巨核球芽細胞株Ｋ５６２細胞を用いて前記（３）から（９）に示す工程を同様に行ない、ＦＡＣＳＡｒｉａにてＮａｎｏｇ陽性とＮａｎｏｇ陰性の境界線を作成した。
Example 1
The difference in cell staining due to the membrane permeation process was evaluated by the following method.
(1) Human iPS cell 201B7 strain (purchased from iPS Academia Japan) was seeded on a petri dish coated with iMatrix (Takara Bio) and cultured in StemFitAK02N medium (Takara Bio).
(2) The iPS cells cultured for 7 days in (1) are washed with PBS (phosphate buffered saline), and then mixed with TrypLE select (manufactured by Thermo Fisher) and Versene Solution (manufactured by Thermo Fisher). Cells were detached.
(3) After recovering the detached cells, the cells were fixed by immersing them in a 4% (w / v) paraformaldehyde solution at room temperature for 40 minutes.
(4) The fixed cells were washed twice with PBS containing 0.5% BSA and 2 mM EDTA (Buffer A) and then replaced with 90% (v / v) methanol under the conditions (b) and (d) on ice. The membrane permeation process was performed by leaving still for 30 minutes.
(5) Buffer A under conditions (a) and (b), 5% (v / v) FBS (fetal calf serum) and 0.75% (w / v) under conditions (c) and (d) Each was washed with PBS (Buffer B) containing saponin.
(6) In conditions (a) and (b), immerse in Buffer A containing rabbit-derived anti-mouse Nanog antibody (manufactured by Cell Signaling Technology), and in conditions (c) and (d), in Buffer B containing the antibody. Each of the cells was immersed for 90 minutes at room temperature to form a complex of an undifferentiated iPS cell and an antibody against the protein (Nanog) of the cell.
(7) Washing was performed twice with Buffer A under the conditions (a) and (b), and with Buffer B under the conditions (c) and (d).
(8) Soaking in Buffer A containing goat-derived anti-rabbit IgG antibody (manufactured by Thermo Fisher) labeled with Alexa Fluor 488, a labeled secondary antibody under the conditions of (a) and (b), (c) and (d ) Were immersed in Buffer B containing the antibody, and reacted at room temperature for 90 minutes.
(9) After washing with Buffer A three times, the ratio of Nanog positive iPS cells was measured with FACSAria (BD Bioscience).
(10) The steps shown in (3) to (9) above are similarly performed using megakaryocyte blast cell line K562 cells as cells that do not express Nanog, and a boundary line between Nanog positive and Nanog negative is created in FACSAria. did.
ＦＡＣＳＡｒｉａの結果を図１に示す。アルコールであるメタノールおよび／または界面活性剤であるサポニンを用いて膜透過処理（（ｂ）から（ｄ）の条件）することで、膜透過処理を行なわなかったとき（（ａ）の条件）と比較し、Ｎａｎｏｇ陽性細胞の割合が向上しており、アルコールまたは界面活性剤により細胞が有する生体膜の脂質成分が可溶化されることがわかる。 The results of FACSAria are shown in FIG. Membrane permeation treatment (conditions (b) to (d)) using methanol as an alcohol and / or saponin as a surfactant (conditions (a)) In comparison, the ratio of Nanog positive cells is improved, and it can be seen that the lipid component of the biological membrane of the cells is solubilized by alcohol or a surfactant.
特にアルコールであるメタノールおよび界面活性剤であるサポニンを用いて膜透過処理したとき（（ｄ）の条件、Ｎａｎｏｇ陽性細胞の割合：８４．４％）は、メタノールまたはサポニンのいずれか一方を用いて膜透過処理したとき（（ｂ）および（ｃ）の条件、Ｎａｎｏｇ陽性細胞の割合：７７．１％および６６．２％）と比較し、Ｎａｎｏｇ陽性細胞の割合がさらに向上していることから、アルコールによる膜透過処理と界面活性剤による膜透過処理とを組み合わせることによって、膜透過処理効率がさらに向上し、抗Ｎａｎｏｇ抗体を含む染色試薬が細胞内へ拡散しやすくなり、細胞中のＮａｎｏｇタンパクとの結合性を改善でき、Ｎａｎｏｇ発現細胞をより高感度に検出できることが示唆される。 In particular, when membrane permeation treatment was performed using methanol as an alcohol and saponin as a surfactant (condition (d), ratio of Nanog positive cells: 84.4%), either methanol or saponin was used. Compared with the membrane permeation treatment (conditions (b) and (c), ratio of Nanog positive cells: 77.1% and 66.2%), the ratio of Nanog positive cells is further improved. By combining the membrane permeation treatment with alcohol and the membrane permeation treatment with a surfactant, the membrane permeation treatment efficiency is further improved, and the staining reagent containing the anti-Nanog antibody is easily diffused into the cell. This suggests that Nanog-expressing cells can be detected with higher sensitivity.
なお、（ａ）から（ｄ）の条件でＮａｎｏｇ陰性細胞（Ｋ５６２株）を測定したところ、いずれの条件もＮａｎｏｇ陽性細胞の割合が１％以下となり、膜透過処理による偽陽性の発生はないことを確認した。 In addition, when Nanog negative cells (K562 strain) were measured under the conditions (a) to (d), the ratio of Nanog positive cells was 1% or less under any condition, and there was no occurrence of false positives due to membrane permeabilization. It was confirmed.
（実施例２）
実施例１（ｂ）および（ｄ）の条件のうち、（４）の膜透過処理で用いるアルコールを９０％（ｖ／ｖ）エタノールとした他は、実施例１と同様な方法で行ない、アルコールによる細胞染色の違いを評価した。
(Example 2)
Of the conditions in Examples 1 (b) and (d), alcohol was used in the same manner as in Example 1 except that the alcohol used in the membrane permeation treatment in (4) was 90% (v / v) ethanol. The difference in cell staining due to was evaluated.
ＦＡＣＳＡｒｉａの結果を図２に示す。膜透過工程で用いるアルコールをエタノールに替えても、実施例１と同様、界面活性剤であるサポニンを用いた膜透過処理を組み合わせる（（ｆ）の条件、Ｎａｎｏｇ陽性細胞の割合：７９．９％）ことによって、エタノールまたはサポニンのいずれか一方を用いて膜透過処理したとき（（ｅ）および（ｃ）の条件、Ｎａｎｏｇ陽性細胞の割合：７３．４％および６６．２％）と比較し、Ｎａｎｏｇ陽性細胞の割合がさらに向上していることから、アルコールによる膜透過処理と界面活性剤による膜透過処理とを組み合わせることによって、膜透過処理効率がさらに向上し、抗Ｎａｎｏｇ抗体を含む染色試薬が細胞内へ拡散しやすくなり、細胞中のＮａｎｏｇタンパクとの結合性を改善でき、Ｎａｎｏｇ発現細胞をより高感度に検出できることが示唆される。 The results of FACSAria are shown in FIG. Even if the alcohol used in the membrane permeation step is changed to ethanol, the membrane permeation treatment using the saponin as a surfactant is combined as in Example 1 (condition (f), ratio of Nanog positive cells: 79.9% ), When compared with membrane permeabilization treatment with either ethanol or saponin (conditions (e) and (c), percentage of Nanog positive cells: 73.4% and 66.2%) Since the ratio of Nanog positive cells is further improved, by combining the membrane permeation treatment with alcohol and the membrane permeation treatment with a surfactant, the membrane permeation treatment efficiency is further improved, and a staining reagent containing an anti-Nanog antibody is obtained. It can easily diffuse into cells, improve the binding with Nanog protein in cells, and detect Nanog-expressing cells with higher sensitivity. There is suggested.
一方、実施例１（ｄ）の条件（Ｎａｎｏｇ陽性細胞の割合：８４．４％）と、本実施例（ｆ）の条件（Ｎａｎｏｇ陽性細胞の割合：７９．９％）とを比較すると、（ｄ）の条件の方がＮａｎｏｇ陽性細胞の割合が高いことから、本発明の染色方法において膜透過工程で用いるアルコールとしてはメタノールが好ましいことがわかる。 On the other hand, when the conditions of Example 1 (d) (Nanog positive cell ratio: 84.4%) and the conditions of this Example (f) (Nanog positive cell ratio: 79.9%) were compared, Since the ratio of Nanog positive cells is higher under the condition d), it is understood that methanol is preferable as the alcohol used in the membrane permeation step in the staining method of the present invention.
なお、（ｅ）および（ｆ）の条件でＮａｎｏｇ陰性細胞（Ｋ５６２株）を測定したところ、いずれの条件もＮａｎｏｇ陽性細胞の割合が１％以下となり、膜透過処理による偽陽性の発生はないことを確認した。 In addition, when Nanog negative cells (K562 strain) were measured under the conditions of (e) and (f), the ratio of Nanog positive cells was 1% or less under any condition, and there was no occurrence of false positives due to membrane permeabilization. It was confirmed.
（実施例３）
実施例１（ｄ）の条件のうち、
（６）の未分化ｉＰＳ細胞−抗Ｎａｎｏｇ抗体複合体形成を、０．５％（ｖ／ｖ）Ｔｒｉｔｏｎ Ｘ−１００（商品名）を含むＰＢＳ（Ｂｕｆｆｅｒ Ｃ）でウサギ由来抗マウスＮａｎｏｇ抗体（セルシグナリングテクノロジー社製）を希釈した反応液に室温で９０分間浸すことで行ない、
（７）の洗浄をＢｕｆｆｅｒ Ｃを用いて行ない、
（８）の標識をＡｌｅｘａ Ｆｌｕｏｒ ４８８で標識したヤギ由来抗ウサギＩｇＧ抗体（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ社製）を含むＢｕｆｆｅｒ Ｃに浸し室温で９０分間反応させることで行なった他は、実施例１と同様な方法で行ない、界面活性剤による細胞染色の違いを評価した。
(Example 3)
Of the conditions of Example 1 (d),
(6) The undifferentiated iPS cell-anti-Nanog antibody complex formation was performed using a rabbit-derived anti-mouse Nanog antibody (cell) in PBS (Buffer C) containing 0.5% (v / v) Triton X-100 (trade name). Signaling Technology) is immersed in a diluted reaction solution at room temperature for 90 minutes,
(7) is cleaned using Buffer C,
The same method as in Example 1 except that the labeling of (8) was carried out by immersing in Buffer C containing goat-derived anti-rabbit IgG antibody (manufactured by Thermo Fisher) labeled with Alexa Fluor 488 for 90 minutes at room temperature. The difference in cell staining with the surfactant was evaluated.
ＦＡＣＳＡｒｉａの結果を図３に示す。アルコールと界面活性剤とを組み合わせて膜透過処理を行なう際、界面活性剤としてＴｒｉｔｏｎ Ｘ−１００（商品名）を用いる（（ｇ）の条件、Ｎａｎｏｇ陽性細胞の割合：７６．２％）と、サポニンを用いたとき（（ｄ）の条件、Ｎａｎｏｇ陽性細胞の割合：８４．４％）と比較し、Ｎａｎｏｇ陽性細胞の割合が低下したことから、本発明の染色方法において膜透過工程で用いる界面活性剤としてはコレステロールを特異的に可溶化するサポニンが好ましいことがわかる。 The results of FACSAria are shown in FIG. When performing membrane permeation treatment by combining alcohol and a surfactant, Triton X-100 (trade name) is used as a surfactant (condition (g), ratio of Nanog positive cells: 76.2%). Compared to the case of using saponin (condition (d), ratio of Nanog positive cells: 84.4%), the ratio of Nanog positive cells was reduced, so that the interface used in the membrane permeation step in the staining method of the present invention It can be seen that saponins that specifically solubilize cholesterol are preferred as the active agent.
なお、（ｇ）の条件での、Ｎａｎｏｇ陰性細胞（Ｋ５６２株）を測定したところ、Ｎａｎｏｇ陽性細胞の割合が１％以下と、膜透過処理による偽陽性の発生はないことを確認した。 In addition, when Nanog negative cells (K562 strain) were measured under the conditions of (g), it was confirmed that the ratio of Nanog positive cells was 1% or less and no false positives were generated by membrane permeabilization.
Claims (6)
（１）固定化試薬を用いて細胞を固定する工程
（２）固定した前記細胞をアルコールを含む試薬で処理することで、前記細胞が有する生体膜の脂質成分を可溶化し透過させる工程
（３）固定した前記細胞を界面活性剤を含む試薬で処理することで、前記細胞が有する生体膜の脂質成分を可溶化し透過させる工程
（４）抗Ｎａｎｏｇ抗体を含む染色試薬を用いて膜透過した前記細胞を染色する工程 A method for staining cells, comprising the steps of (1) to (4) below.
(1) Step of fixing cells using an immobilization reagent (2) Step of solubilizing and permeating lipid components of biological membranes of the cells by treating the immobilized cells with a reagent containing alcohol (3 ) Step of solubilizing and permeating the lipid component of the biological membrane of the cell by treating the fixed cell with a reagent containing a surfactant (4) Permeating the membrane using a staining reagent containing an anti-Nanog antibody Staining the cells
（１）ホルムアルデヒドまたはパラホルムアルデヒドを含む固定試薬
（２）メタノールを含む生体膜透過試薬
（３）サポニンを含む生体膜透過試薬
（４）抗Ｎａｎｏｇ抗体を含む染色試薬 A kit comprising at least the reagent according to the following (1) to (4).
(1) Fixing reagent containing formaldehyde or paraformaldehyde (2) Biomembrane permeation reagent containing methanol (3) Biomembrane permeation reagent containing saponin (4) Staining reagent containing anti-Nanog antibody
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