细胞分选 Hello every one. I am glad to be here and have this opportunity to give a talk about our StemCell Technologies company and our company’s products

细胞分选和基本原理 细胞分选是把一种细胞从多细胞样品中分离出来 用抗体标记细胞，同时被用的抗体被萤光标记或连接免疫磁珠或其它 利用萤光和免疫磁珠的特性来分离细胞 Before we go into details of our products, let’s get it clear: what is cell separation and how to do it? Most samples such as blood and bone marrow are cell mixture. To study one type cell function, we need isolated the cell out of the mixture. This approach is cell separation. To separate cells, we usually label cells with antibody. Meanwhile, the antibody is labeled with fluorescence molecule or linked to a particle or some sort…, Then use fluorescence or particles to separate cells.

细胞分选常用方法 密度梯度离心(Ficoll)离心 抗体标记分离 免疫密度离心 免疫磁珠 流式

正选 和 负选 正选 负选 标记需要得细胞 标记不需要得细胞 需要得细胞 需要得细胞 正选 和 负选 正选 需要得细胞 标记需要得细胞 需要得细胞 标记不需要得细胞 负选 two basic approaches in cell separation, which are positive selection and negative selection.

正选和负选的比较 优点 缺点 正选 只需要一种抗体 分离出的细胞纯度高 对于比例小的细胞群，正选容易得到高纯度 负选 需要的细胞没有被抗体等标记 对需要的细胞，不需要特异性的细胞标记 优点 缺点 需要特异性的靶细胞标记 可能引起细胞活化 需要多种抗体标记不需要的细胞

细胞分选产品 Today I am going to focused on our sell separation productions. As you see here, we have several sell separation systems for you to choose. With these products, we can separate human cell, mouse, rat and rhesus monkey cells.

StemCell Technologies Inc. 的细胞分选原理 利用抗体四聚体(Tetrameric Antibody Complex, TAC)来分选细胞 TAC Cell 磁珠 直接标记 TAC Cell 磁珠 间接标记 StemCell technologies has a unique way to separate cells. That is TAC technology. This graph show the TAC structure which is two mouse antibodies are cross linked by rat anti-mouse antibodies. In this case, one mouse antibody targets cell surface marker and the other binds to particle. This is called direct labeling. Another is called indirect labeling. That is cells are linked with labeled antibody, can be biotin or fluorescence labeled antibody and then use anti biotin or fluorescence TAC. Using the TAC technology, we developed our cell separation products.

适用于人的全血细胞样品 是一种负选方法 基本原理：抗体标记和免疫密度离心法 需要的仪器：离心机 By using our TAC technology, we developed following cell separation systems. RosetteSep is suitable for human whole blood sample It’s a negative selection Basic approach include cell labeling and density centrifugation Only need centrifuge

Direct Labeling of Human Cells 不需要的细胞 The mechanism behind this is we use two types of TAC. One target unwanted cells and link to red blood cells, the other cross link red blood cells. After spin, unwanted cells will be pellet with red blood cells.

分选的过程 标记 离心 收集 室温下20分钟 用缓冲液稀释 把RosetteSep™ 抗体混合液加入细胞样品中 把细胞样品置于密度分离液上 The separation procedure is very simple. Add antibody cocktail, incubate, layer the sample on the density medium, and spin. After spin, cell is ready to collect.

的密度分离液DM-L 为了最大限度回收淋巴细胞，StemCell专门研制了一种适合RosetteSep™的密度分离液—— DM-L DM-L 的密度：1.081 g / mL DM-L 的渗透压与人外周血的渗透压相同(iso-osmolar) 适用于以下的淋巴细胞分选: CD3+ T cells - CD8+ T cells CD4+ T cells - B cells To maximize lymphocyte recovery, we recommended to use our DM-L density medium. DM-L density is 1.081 and has the same osmolarity as peripheral blood It is recommended for RosetteSep T an B lymphocyte separation. The reason for that is

From: “Centrifugation Techniques III”, Nycomed Amersham Density of Fresh Leukocytes Monocytes Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes DM-L: 1.081 g/mL Ficoll: 1.077 g/mL RosetteSep is based on density centrifugation, most used the density medium Ficoll density is 1.077. All the cells which density are higher than 1.077 will be spin down. This will include part of the lymphocytes. Therefore use DM-L will obtain maximum lymphocyte recovery. 1.060 1.070 1.080 1.090 1.100 Density in g/mL From: “Centrifugation Techniques III”, Nycomed Amersham

人 和 小鼠 低温冷藏的外周血单个核细胞 脾细胞 人 和 小鼠 低温冷藏的外周血单个核细胞 脾细胞 After RosetteSep launched, a lot of our customer asked us if you have a similar products for mouse spleen and human previous frozen PBMC. Because the method is so simple and easy. For this purpose, we developed SpinSep.

的特点 Sep 负选, 只需要离心机 可以同时分选多份样品 分选的细胞量在 适用于低温冷藏的人外周血单个核细胞(PBMC)和小鼠脾细胞 取决于离心机的容量 分选的细胞量在 106 cells in Eppendorf - 109 cells in 50 mL tube 适用于低温冷藏的人外周血单个核细胞(PBMC)和小鼠脾细胞 Again, SinSep is a negative selection and only need centrifuge It can process multi-samples depending on your centrifuge capacity It can process as little as 10^6 cell per sample up to 10^9 cells This approach is designed for previous frozen PBMCs and mouse spleen cells.

直接标记人的细胞 Anti-cell Anti- particle Dense Particle(1mm) Unwanted Cell Dense Particle(1mm) This diagram shows SpinSep direct label human cells through TAC and link to SpinSep dense particles. Tetrameric Antibody Complex (TAC)

间接标记小鼠的细胞 Biotinylated Antibody Anti- Anti- Biotin Particle Unwanted Cell SpinSep indirect label mouse cells through anti-biotin TAC. You may notice SpinSep dense particles are relatively bigger about 1um diameter. Tetrameric Antibody Complex (TAC) Dense Particle(1mm)

SpinSep 的分选过程 Label Layer & Spin Collect Antibody Cocktail Anti-Biotin TAC (mouse only) Dense Particles 4. Layer over SpinSep® Density Medium Spin 10-20min at 1200xg Collect almost entire volume 7. Wash to remove Density Medium SpinSep procedure is very simple. Basically add antibody cocktail, incubate, and layer the sample over SpinSep Density Medium. Then Spin and collect your cells from interface. One important thing is that SpinSep Density Medium is special medium only for SpinSep. In other words, you cannot use any other density medium to replace. Centrifugation speed? Collect entire volume?

的细胞分选资料 人的外周血细胞(PBMC) 小鼠的脾细胞 Values are the mean ± SD Here is SpinSep performance data for you reference. Again this is negative selection approach. The purity both in human and mouse cells are very good. Values are the mean ± SD

新：不用柱子，只需用磁极，所用的磁珠很小 高效：效率-分选过程不用洗细胞(省时省力); 效果-分选的细胞纯度高 易：操作过程简单易行 新型，高效，易行的免疫磁珠分选技术 新：不用柱子，只需用磁极，所用的磁珠很小 高效：效率-分选过程不用洗细胞(省时省力); 效果-分选的细胞纯度高 易：操作过程简单易行 广：可做正选和负选 适应性强：在理论上可以分选任何一种细胞 As you see, StemSep has excellent performance data. However, it required column just like most of other companies’ cell separation products. The function of column is to enhance the magnetic field which is required for catching the tiny particles. To simplify separation procedure, our scientists developed a new cell separation system. EasySep- is a new, highly efficient and easy magnetic separation technique - EasySep. It’s new because it’s column free. Only use magnet. This magnet is different from others. It generates a very strong magnetic field. It’s highly efficient because the separation is non-wash procedure, save you a lot of time. The performance is excellent The operation is very easy Broad application: it is used for positive selection and negative selection. Very flexible: theoretically, it can separate any kind of cells.

Magnetic Nanoparticle 直接标记人的细胞 Anti- Anti- Cell Particle Cell Tetrameric Antibody Complex (TAC) Magnetic Nanoparticle (160 nm)

Magnetic Nanoparticle 间接标记小鼠细胞 Biotin/Fluo Antibody Anti- Anti- biotin/Fluo Particle Cell Tetrameric Antibody Complex (TAC) Magnetic Nanoparticle (160 nm)

Relative Sizes of Magnetic Particles used in Cell Separation Our EasySep nanoparticles are a little bigger that StemSep particles. But it doesn’t affect flowcytometry data analysis.

的细胞分选过程 标记 结合磁珠 分选 Ab (TAC) (人) Abbiotin anti-biotin TAC(小鼠) 加入免疫磁珠 孵育后稀释 放置磁极中 倒出未标记的细胞 分选 The separation procedure is very easy and simple. Add antibody or antibody cocktail, incubate, and add nanoparticles. After incubation, place the tube in magnet for 5-10 min separation. Finally pour off unlabeled cells.

正选资料 人的外周血细胞(PBMC) 小鼠脾或骨髓细胞 What Lin-/ c-Kit+/Sca 1+ 4.3? Mouse T CD90, why don’t choose CD3?

负选资料 人的外周血细胞(PBMC) 小鼠脾细胞

可正选或去除任何一种细胞 PE 标记的抗体分选Kit FITC 标记的抗体分选Kit Biotin 标记的抗体分选Kit Cell EasySep is very flexible. Technically, it can separate any kind of cells. We provide PE, FITC and biotin selection TAC. As long as you have PE, FITC or biotin labeled antibody. You can separate the cells.

细胞分选常见的问题 分选方法的选择 样品种类 样品数量 每次需要分选的细胞数

常见问题 样品的前处理 Ficoll 离心机 全血和单采的细胞 细胞计数 稀释液 细胞数

常见问题 分选过程中 细胞浓度 缓冲液 细胞混匀动作要点

Calculating Purity and Recovery % Purity = # desired cells in enriched fraction x 100 # total nucleated cells in enriched fraction = % of total events in the desired cell gate (FACS profile) % Recovery = # desired cells in enriched fraction x 100 # desired cells in start = (% purity in enriched) x (total nucleated cells in enriched) x 100 (% purity in start) x (total nucleated cells in start)

FACS Gate

panNK+ Selected from Mo Spleen Start NK+ Selected

Data analysis

的技术要领 细胞稀释后，用吸管混匀细胞（不要倒置试管） 倒出未标记的细胞时，让液体连续流淌，试管停留在最后的位置约2秒钟，但不要刻意抖下最后一滴细胞液 Technical tips When mixing, pipette up and down gently. Don’t invert tube. Pour cell off gently in a smooth motion, Leave tube in invert position for 2 seconds, but don’t shake or blow off last drop. Cells can be poured into a large tube and transferred into 5 ml FACS tube for subsequently separation.