从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的
Summary
This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.
Abstract
Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.
Introduction
皮肤状况，从接触性皮炎，湿疹，牛皮癣，蜂窝组织炎，真菌感染和脓肿，以非黑色素瘤皮肤癌被认为是世界范围内的50个最流行 的疾病之一，非致命疾病于2010年1第四全球领先的原因。因此，底层的多样皮肤病理学分子和细胞机制的调查是研究的一个必要的和活跃的领域。啮齿动物模型已在炎性皮肤状况的了解非常有用的，例如特应性皮炎2，牛皮癣3，或金黄色葡萄球菌感染 4。对于小鼠皮肤组织的酶消化廉价，高效，且简单的协议可以提供细胞，它可以用于各种下游应用，以便更好地了解皮肤疾病的病理生理的制剂。这里，一个简单而经济的方法被描述为小鼠皮肤的酶消化组织和皮肤浸润白细胞的隔离，可以用来进行细胞培养， 在体内继转移，流式细胞仪分析和分选或基因表达研究。此过程的总的目标是制备皮肤浸润白细胞单细胞悬浮液与高细胞生存力，同时尽量减少通常与定制试剂盒和机械dissociators相关的成本。
现有的皮肤组织裂解方法5-7可能导致低细胞活力及表面标志的完整性，或需要定制酶试剂盒和昂贵的组织分离机8-11。而鼠耳皮肤组织的消化是合理流行12-13消化高度角化皮肤组织（例如，从侧面）可导致污染大量非细胞碎片的细胞制剂。在最近的一项研究中，扎伊德和同事在2.5毫克消化鼠标侧面的皮肤90分钟/ ml的分散酶，弗洛45分钟，在3毫克/毫升胶原酶7结婚。在另一项研究中，这些研究人员使用的多个孵育，用2.5小时的结合消化，包括使用胰蛋白酶/ EDTA，胶原酶III的和分散酶5。不推荐用于酶促消化皮肤使用胰蛋白酶，与来自不同制造商的胰蛋白酶治疗已经显示出可测量的影响对哺乳动物细胞14-15的细胞表面蛋白的完整性。此外，分散酶可以对CD4和CD8αT细胞增殖的能力显著作用和影响的至少20分子的表面丰度，包括常见的T细胞活化标记物如CD62L 16。其他协议使用RPMI 1640在消化介质 6。然而， 镁离子和钙离子的在RPMI的存在会引起广泛的细胞聚集17。
一个理想的协议，用于组织分离的目标应该是高细胞活力，低细胞聚集，和损害最小的水平与细胞表面蛋白。高质量淋巴结基质细胞的准备工作已经完成，从而使用较短的酶孵育，钙2+和Mg 2+免费媒体协议，并避免胰蛋白酶和分散酶18。然而，这种类型的协议尚未建立用于全鼠皮肤的解离。
这里，一个协议描述以解离，隔离和从过敏原挑战小鼠侧翼皮肤丰富皮肤浸润白细胞。简言之，将切下的皮肤是预孵育中Hank氏平衡盐溶液（HBSS）中，用10％胎牛血清1小时，以软化组织的消化和除去任何过量的死皮或脂肪组织。这之后是30分钟的酶消化步骤与0.7毫克/毫升胶原酶D.胶原酶D具有对细胞表面标记物密度的影响最小，并在体外 16,18对T细胞增殖没有影响，使得它非常适用于涉及表面蛋白的表征的应用程序。下列酶消化，不连续密度梯度离心法从单细胞悬浮液除去上皮细胞和碎片和富集造血细胞。重要的是，此过程避免了昂贵的基于列的磁性细胞分离试剂和组织离解机8-11，并且可以与设备和材料在碱性生物医学研究实验室发现来执行。此处这个协议是用于分离白细胞从侧翼皮肤挑战三次半抗原恶唑酮（牛）在先前致敏ND4 Swiss小鼠（改编自19）。使用多参数流式细胞仪分析细胞。这种技术产生了细胞悬浮液以最小的碎片和分离的淋巴细胞的> 95％的存活率这是由多参数流式细胞术分析来测量的T淋巴细胞和嗜中性粒细胞渗入影响SK在。
Protocol
Representative Results
Discussion
表征变化，皮肤的居民白细胞皮肤疾病的啮齿动物模型，如特应性皮炎或银屑病是理解炎症细胞大量涌入和疾病病理之间的机械连接非常重要。在这里，我们描述了一个经济的技术来隔离皮肤组织的白细胞在大多数生物医学研究实验室发现基本设备。这种较快的技术避免了使用昂贵的组织分离机和自管和试剂，有助于节约资源，同时尽量减少手工操作时间在实验室工作台上。温和酶促消化和不连?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
美国国立卫生研究院（NIH R15 NS067536-01A1直流），国家慢性阴部疼痛协会（奖DC），以及麦卡莱斯特学院支持这项工作。 CB获得了奖学金从麦卡利斯特大学贝克曼学者计划，由阿诺德和梅布尔贝克曼基金会资助。 BTF和TM是由JDRF 2-2011-662支持。我们感谢贾森申克尔博士和朱丽安娜·刘易斯博士的技术咨询，以及Chatterjea实验室的所有现任和前任成员的帮助和支持。
Materials
| HBSS with phenol red, without calcium, without magnesium, liquid | Sigma Aldrich | 55021C | Keep sterile until day of usage. |
| HEPES, ≥99.5% (titration) | Sigma Aldrich | H3375 | |
| EDTA disodium salt solution | Sigma Aldrich | E7889 | Keep sterile until day of usage. |
| Fetal Bovine Serum, USDA, Heat Inactivated, Premium Select | MidSci | S01520HI | Keep sterile until day of usage. |
| Percoll, pH 8.5-9.5 (25°C) | Sigma Aldrich | P1644 | Keep sterile. Percoll needs to be made isotonic with sterile 10X PBS prior to use. |
| Trimmer Combo Kit | Kent Scientific | CL9990-1201 | Use the larger trimmer for shaving the flank and back. |
| 4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one | Sigma Aldrich | E0753-10G | Dissolve in 100% EtOH at 50°C for 15 minutes on a rotating plate. |
| Purified anti-mouse CD16/CD32 (2.4G2) | Tonbo Biosciences | 70-0161 | Antibody used to block non-specific antibody binding during antibody staining for flow cytometry |
| anti-CD3e-BV650 (145-2C11) | Becton Dickinson | 564378 | Antibody used to stain cells for flow cytometry |
| anti-CD4-APC-eFluor780 (RM4-5) | eBioscience | 47-0042-80 | Antibody used to stain cells for flow cytometry |
| anti-CD8a-BV785 (53-6.7) | BioLegend | 100749 | Antibody used to stain cells for flow cytometry |
| anti-CD11b-eFluor450 (M1/70) | eBioscience | 48-0112-80 | Antibody used to stain cells for flow cytometry |
| anti-CD11c-eFluor450 (N418) | eBioscience | 48-0114-80 | Antibody used to stain cells for flow cytometry |
| anti-CD44-BV711 (IM7) | Becton Dickinson | 563971 | Antibody used to stain cells for flow cytometry |
| anti-CD45-FITC (30-F11) | Tonbo Biosciences | 35-0451-U025 | Antibody used to stain cells for flow cytometry |
| anti-CD45R-eFluor450 (RA3-6B2) | eBioscience | 48-0452-80 | Antibody used to stain cells for flow cytometry |
| anti-CD62L-PerCP-Cy5.5 (MEL-14) | BioLegend | 104431 | Antibody used to stain cells for flow cytometry |
| anti-Ly-6G/Ly-6C (Gr-1)-PE (RB6-8C5) | BioLegend | 108407 | Antibody used to stain cells for flow cytometry |
| Ghost Dye-BV510 | Tonbo Biosciences | 13-0870-T100 | Viability dye for flow cytometry |
| LSR Fortessa | Becton Dickinson | N/A | Cell analyzer (18 parameters) |
| Collagenase D from Clostridium histolyticum | Roche Applied Science | 11088858001 | Aliquot lyophilized enzyme at 5 mg/mL in HBSS with phenol red, without calcium, and without magnesium into 1 mL aliquots. Store immediately at -20°C for up to six months. |
| ND4 Swiss female mice | Harlan | 0-32 | 8-12 weeks old; conventionally housed with free access to food and water and used according to Macalester College's IACUC guidelines. |
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