stemcell新品：EasySep™人 CD45阳选试剂盒（磁珠可解离）

作为STEMCELL代理商，嘉远生物很高兴宣布其新产品：EasySep Release Human CD45 Positive Selection Kit。文末全是干货，包含了各种组织解离方法，你值得拥有！

产品信息：

该产品适用于从人原代组织和肿瘤组织（例如肿瘤浸润性白细胞[TIL]）的单细胞悬液或人源化小鼠的组织和异种移植肿瘤中分离CD45 +白细胞。

产品优势：

无堵塞。无柱系统消除了贵重样品发生堵塞从而损失细胞的风险。

灵活性。有适用于多种样本的方案，如人和人源化小鼠的正常组织和肿瘤组织。

细胞无磁珠。细胞分选后可以进一步释放磁珠，从而得到无磁珠的阳选细胞。这些细胞可立即用于进一步的免疫细胞亚群分离，下游分析或培养。

快速高效。简单一倒就能获得白细胞的41分钟操作方案。

产品数据：

Figure 1. EasySep™ Release Human CD45 Positive Selection Kit

Starting with a single-cell suspension of a human breast cancer tumor xenograft (MDA-MB-231) sample from an NRG-3GS humanized mouse, the CD45+ TIL purities of the start and final isolated fractions are 5.5% and 96.0%, respectively.

NOTE: Cell debris and dead cells were excluded from the analysis based on DRAQ5™ and DAPI fluorescence.

Figure 2. EasySep™-Isolated CD45+ Cells are Representative of the Starting Leukocyte Population

Mass cytometry data comparing the composition of immune subsets in PBMCs and EasySep™-isolated cells from the same donor. Starting with whole blood, PBMCs were prepared by density gradient centrifugation using Lymphoprep™. To compare immune subset composition pre- and post-EasySep™ isolation, a fraction of the PBMCs was further isolated using EasySep™ Release Human CD45 Positive Selection Kit and the pre- and post-isolated fractions were assessed by mass cytometry (CyTOF®). t-SNE plots of cells stained with 19 markers and analyzed by CyTOF® are shown (n = 1).

Figure 3. EasySep™-Isolated CD45+ Cells Produce IFN-gamma in Response to Antigen and Mitogen Stimulation

PBMCs pre- and post-EasySep™ isolation were incubated for 24 hours in the presence of peptide pools (CEF for antigen-specific CD8+ T cell response and CPI for antigen-specific CD4+ T cell response) or mitogen (phytohemagglutinin [PHA]). Following incubation, ELISpot plates were processed and IFN-gamma-producing cells were counted using an AID ELISpot reader. Representative images of ELISpot assays are shown (n = 3).

Figure 4. CD45+ Cells Isolated by EasySep™ from Various Tissues Are Highly Purified

A humanized mouse tumor model was generated by engraftment of human CD34+ hematopoietic stem and progenitor cells into NRG-3GS mice followed by xenotransplantation with human cancer cell lines, MDA-MB-231 (breast cancer) or SKOV-3 (ovarian carcinoma). Starting with a single-cell suspension of spleens, lungs, bone marrow, or tumors, human CD45+ leukocytes were isolated using EasySep™ Release Human CD45 Positive Selection Kit. The starting frequencies and isolated purities of human CD45+ cells of individual experiments and averages are shown.

下游应用：

流式检测或分选 (免疫表型和/或亚型分选)

RNA 测序 (包括单细胞测序如10X Genomics)

细胞因子分析

基因组分析

细胞培养 (包括扩增和共培养)

相关产品：

技术资料：

组织解离方法指南：

小鼠脾脏机械解离步骤

此实验步骤概述了如何从脾脏样本中收集细胞，并制备成单细胞悬液以进行下游细胞分选。从原代组织样本中制备真正的单细胞悬浮液可避免过量的细胞损失并且能够最大化标记靶细胞，从而优化细胞分选。

实验步骤

1.将脾脏组织转移到35 mm无菌培养皿（产品号 #27100/27150）中。后续如需进行细胞分选，在培养皿中加入5 mL推荐培养基（可请请参阅试剂盒产品说明书）。如解离组织后不需细胞分选，请在培养皿中加入含1 mM EDTA的PBS。

a.注意: 如同时处理多个脾脏样本，需使用100 mm培养皿（产品号 #27110），并加入10 mL培养基。

2.去除脾脏组织上的结缔组织或脂肪，注意坏死区域，并检查脾脏是否有肿大、变色或病变。记录起始样本的状态对于后期评估细胞非常重要。

3.从3cc无菌注射器（产品号 #28230/28240）中取出活塞/柱塞。使用扁平的末端部分以温和画圆的方式研磨脾脏5次。这样可以破坏脾脏和髓质，释放脾细胞。

4.将一个70 μm细胞过滤器（产品号 #27216/27260）置于50 mL无菌锥形管上，并用2 mL推荐培养基润洗滤网。

a.注意：细胞可能会黏附于干燥的滤网上，润洗滤网可以减少这种情况 。

5.使用润洗过的5 mL或10 mL移液管将脾细胞吹打均匀。

6.将上清液和组织从培养皿转移至润洗过的70μm细胞过滤器（产品号 #27216/27260）。使用一个新的3cc无菌注射器的活塞/柱塞，以温和画圆的方式使细胞和组织通过滤网。用3 mL推荐培养基冲洗滤网。

a.如需提高回收率，可选择进行此步骤：

i：只将上清液而不要将脾脏组织移入过滤器。在培养皿中另加入5 mL推荐培养基。注意：如果一次处理多个脾脏可加入10 mL推荐培养基。

ii：重复步骤4-6，在培养皿中加入推荐培养基（5 mL或10 mL）直至脾脏组织变白且培养基呈现澄清状态。

7.使用3 mL推荐培养基再次冲洗滤网。

8.离心300 x g，10分钟来收集细胞。

9.小心弃去上清。

10.轻轻敲击试管重悬细胞。

11.如有需要，可在试管中加入40 mL推荐培养基，进行第二次洗涤。重复步骤8-10。

12.脾细胞可用于下游应用。