作者感谢 Delwel 教授和 Touw 教授为我们提供了32维/克脑脊液 R 细胞线, 丹尼尔教授 Tenen 为我们提供了 Bosc23 细胞系。这项工作得到了捷克共和国赠款机构 (GACR 15-03796S 和 GACR 17-02177S) 赠款的支助, 由捷克科学院分子遗传学研究所 (RVO 68378050) 提供的支助, 以 ma j, 一项 GA 英国研究金 (341015 号项目)。从布拉格的查尔斯大学到 MK, 和一个 GA 英国奖学金 (1278217 号项目) 从布拉格查尔斯大学到 PD。

注: 以下是描述32维/克-脑脊液 R 细胞的扩张、分化和转导的步骤。
1. 准备
<ol><li>媒体准备
	<ol>
<li>准备250毫升培养基: RPMI (罗斯威尔公园纪念研究所) 1640 中辅以10% 种热灭活 (胎牛血清) 和小鼠 IL-3 (10 ng/毫升)。<ol>
<li>或者, 使用自制的 IL-3。生产国产 IL-3, 传感器 HEK293 细胞 IL-3 表达载体和收集 IL-3 含有上清22。 注: 抗生素, 如青霉素 G (100 毫升/ml), 链霉素 (100 µg/毫升), 和庆大霉素 (40 µg/毫升), 可用于细胞培养在任何步骤的议定书, 除非另有说明。</li>
		</ol>
</li>
		<li>准备50毫升的分化培养基: RPMI 1640 培养基辅以10% 的血清, 人的 G 脑脊液 (100 ng/毫升)。 注: (重要) 并非所有血清批次都支持32维/克-脑脊液 R 细胞的分化。实验开始之前, 测试不同批次的血清。建议测试至少4种不同的批次, 并选择最佳的基础上的能力, 32 维/克脑脊液 R 细胞区分。请参阅下面的32维/克-CSF 鉴别协议。</li>
		<li>准备10毫升的冷冻培养基: RPMI 1640 培养基辅以40% 的血清, 10% 二甲基亚砜。</li>
	</ol></li>
	<li>逆转录病毒的生产
	<ol>
<li>将 Bosc23 细胞 (6 x 106) 的单个细胞悬浮在16厘米培养皿中, 并在18毫升的 DMEM (Dulbecco 的修饰鹰的培养基) 中培养, 其中含有10% 个血清, 直到培养的融合达到 80% (24 h)。 注意: 细胞应该以单层生长, 而不是在培养中形成团簇。可以使用不同的方法执行单元格计数 (对此处提供的其他协议有效)。在样品数量较少的情况下, 建议使用 Bürker 室在显微镜下进行手动计数。在这种情况下, 使用台盼蓝来排除死细胞。混合单元1:1 与0.4% 台盼蓝在 PBS。要对大量样品进行计数, 可以使用自动单元计数器。</li>
		<li>结合40µg 的逆转录病毒构造 (如 MSCV), 20 µg 的 pCL-生态 (包装载体)23, 80 µl 的裴 (聚乙烯亚胺) 和2毫升的减少血清培养基 (如, 无抗生素)。在室温下 (RT) 孵育这种混合物20分钟。</li>
		<li>小心地取代 Bosc23 细胞培养基与16毫升 DMEM 补充2% 的血清。预热中至37°c 使用前。 注: 在转染过程中不要使用抗生素, 因为它可以降低转染效率。</li>
		<li>添加1.2.2 中制备的混合物。滴状和仔细的 Bosc23 文化, 并孵化4小时在37摄氏度。由于裴毒性, 限制孵化到少于6小时。</li>
		<li>孵化后, 改变 Bosc23 培养基至18毫升预热 DMEM, 含10% 的血清, 并培养48小时在37摄氏度的细胞。将菜肴放在生物安全2级实验室, 从这一步骤保持在这里, 并遵循标准的安全程序。</li>
		<li>收集 Bosc23 培养基 (含有 ecotropic 逆转录病毒粒子), 使用25毫升血清吸管进入50毫升圆锥管, 并存储在4摄氏度。 注: (重要) 转染效率应达到 90%, 以产生高效价病毒。</li>
		<li>加入18毫升预热 DMEM 10% Bosc23 细胞, 培养24小时。</li>
		<li>重复步骤1.2.6。 注: 1.2.6 和1.2.8 的逆转录病毒上清液, 一旦达到相同的温度 (4 摄氏度), 就可以进行汇集, 以保持病毒的完整性。</li>
		<li>为了避免病毒上清液中的 Bosc23 污染, 在4摄氏度时, 将收集到的病毒在 1500 x g 处旋转10分钟。整除病毒, 用干冰或液氮冷冻, 并贮存在-80 摄氏度。然而, 请注意, 新制备的病毒在感染效率方面比冷冻储存的质量高。 注意: (重要) 避免病毒重复冰冻/解冻, 因为它导致病毒的降解。</li>
	</ol></li>
	<li>慢病毒北疆生产
	<ol>
<li>单细胞悬浮 HEK293T (人胚肾 293T) 细胞 (6 x 106) 在 16 cm 培养皿和培养在18毫升 DMEM 包含10% 个血清, 直到文化的融合达到 80% (24 h)。 注意: 细胞应该以单层生长, 而不是在培养中形成团簇。</li>
		<li>结合15µg 的慢病毒载体构造 (如 pGhU6), 包装质粒 pCMVdR8.74 (编码为插科打诨/波兰, 12 µg) 和 pMD2. VSVG (编码为 VSV G, 1.4 µg), 85.2 µL 裴, 2 毫升的减少血清培养基 (没有抗生素)。在 RT 中孵化这种混合物20分钟。</li>
		<li>小心地更换 HEK293T 培养基与16毫升的 DMEM 补充2% 的血清。预热中至37°c 使用前。在转染过程中不要使用抗生素, 因为它可以降低转染效率。</li>
		<li>小心地将1.3.2 中制备的混合物放到 HEK293T 细胞培养中, 在37摄氏度孵化4小时。将潜伏期限制在6小时以内以防止裴毒性。</li>
		<li>改变培养基到18毫升预热 DMEM 10%, 培养细胞48小时在37摄氏度。将菜肴放在生物安全2级实验室, 从这一步骤保持在这里, 并遵循标准的安全程序。</li>
		<li>收集 HEK293T 介质 (含有 amphotropic 慢病毒载体粒子), 使用25毫升血清吸管进入50毫升圆锥管, 并将其存储在4摄氏度。 注: (重要) 转染效率应达到 90%, 以产生高效价病毒。</li>
		<li>小心地添加18毫升预热 DMEM 10% 的 HEK293T 细胞。在37摄氏度培养24小时。</li>
		<li>重复步骤1.3.6。 注: 慢病毒载体上清液从1.3.6 和1.3.8 可以汇集, 一旦他们达到相同的温度 (4 °c), 以保持病毒的完整性。</li>
		<li>为了避免病毒上清的 HEK293T 细胞的污染, 旋转收集的病毒在 1500 x g 10 分钟4摄氏度。整除病毒, 用干冰或液氮冷冻, 并储存在-80 摄氏度。但是, 请注意, 新制备的病毒在感染效率方面比冷冻储存的质量高。 注意: (重要) 避免病毒重复冰冻/解冻, 因为它导致病毒的降解。</li>
	</ol></li>
	<li>含有 GFP 报告的病毒滴定法
	<ol>
<li>种子 1 x 105 NIH/3T3 细胞在300µL 的 DMEM 10% 的血清, 每井到24井板。7口井将被用来效价一种病毒。</li>
		<li>解冻病毒在37°c, 并添加 1, 5, 10, 50, 100, 500 µL 病毒的 NIH/3T3 文化。不要将任何病毒添加到负控制好。培养细胞在病毒存在48小时在37摄氏度。</li>
		<li>收获 NIH/3T3 细胞使用30µL 胰蛋白酶/EDTA (乙二胺二乙酸) (0.25% 胰蛋白酶, 0.01% EDTA 在 PBS) 和放置细胞在250µL PBS 在一个外地活动 (荧光活化细胞分拣) 管。</li>
		<li>通过流式细胞术在每个示例24中确定 GFP+单元格的频率。通过添加活性染料 (如赫斯特 33258) 排除死细胞。</li>
		<li>计算 gfp+单元格的总数 (根据被镀的单元格数和 gfp+单元格的百分比), 并将它们与用于感染的病毒量进行绘制。 注: (重要) 当应用大剂量病毒时, 感染的效率达到高原。因此, 重要的是要估计的效价基于病毒浓度的感染效率发生在一个线性范围 (如表 1中所示)。GFP+单元格的数量指示特定病毒卷中功能性病毒粒子 (TU 转换单位) 的数量。重新计算每毫升的土数。</li>
	</ol></li>
	<li>含有嘌呤霉素报告的病毒滴定法
	<ol>
<li>种子 5 x 104 NIH/3T3 细胞在3毫升的 DMEM 10%, 每井在6井板;使用6口井来滴出一种病毒。文化隔夜在37°c。</li>
		<li>第二天稀释病毒, 如表 2所示。为了稀释病毒, 使用预热后的 DMEM 10% 的血清。不要旋涡。</li>
		<li>从 NIH/3T3 细胞中去除培养基, 加入1毫升稀释病毒。</li>
		<li>孵化过夜在37摄氏度。</li>
		<li>用2毫升预热的 DMEM 10% 的血清, 轻轻地取代含有病毒的培养基, 并在37摄氏度一夜之间孵化。</li>
		<li>轻轻地取代 NIH/3T3 培养基与2毫升预热 DMEM 10% 血清含嘌呤霉素 (2 µg/毫升)。</li>
		<li>文化在37°c 和仔细刷新培养基与嘌呤霉素每3天或当中等变成黄色。</li>
		<li>感染后10-12 天, 从各井中抽出培养基, 用 PBS 轻轻冲洗。</li>
		<li>着色与1毫升水晶紫罗兰溶液 (0.5% 水晶紫在20% 乙醇和 dH2O), 2 分钟在 RT, 并且仔细洗涤与 PBS 两次。</li>
		<li>计数蓝色殖民地出现在每个井在显微镜之下 (放大 X4)。在负控制井中不应观察到菌落。</li>
		<li>计算考虑稀释因子的土/毫升的总数量。</li>
	</ol></li>
</ol>2. 扩展和维护32维/克-脑脊液 R 细胞
<ol><li>如果32维/克-脑脊液 R 细胞被冷冻, 采取一个整除和解冻细胞在37°c 水浴1分钟, 并将细胞从瓶子直接倒入10毫升的预预热 RPMI 1640 培养基补充10% 毫升的管。反转管3次, 旋转细胞向下 400 x g. 在含有培养基的 IL-3 中移除上清和并用重悬细胞, 浓度为 0.3 x10 6 细胞/毫升。</li>
	<li>最佳细胞生长浓度为 0.25-0. 5 x 106细胞/毫升。每2天拆分单元格, 使其浓度为 0.1-0.2 x 106单元格/毫升。 注: (重要) 32 维/克-脑脊液 R 细胞可能部分丧失其分化的能力, 如果生长到浓度高于 1 x 106细胞/毫升。因此, 在时间上分裂32维/克-脑脊液 R 细胞是非常重要的。</li>
	<li>根据需要, 冷冻和储存细胞整除数长时间在-80 °c 或液态氮。向下旋转 3 x 106单元格, 移除上清, 并在1毫升的冷冻培养基中并用重悬。把管子放在冷冻容器里-80 摄氏度。对于长期存储, 将单元格重定位到液氮。</li>
</ol>3. 转导32维/克-脑脊液 R 细胞
<ol><li>在 0.3 x 106细胞/毫升浓度下, 将32维/克脑脊液 R 细胞转化为6井板。</li>
	<li>添加适当数量的病毒达到10和40之间的语言 (感染多样性)。 注意: 更高的教学语言将导致 GFP+细胞的百分比增加, 以及感兴趣基因表达的更高层次。示例: 感染 0.3 x 106细胞, 其语言为 10;3 x 106 TU 将需要添加到32维/克-脑脊液 R 细胞。</li>
	<li>添加凝聚胺到最终浓度8µg/毫升和孵化6小时37摄氏度。或者, 执行一个自旋接种过程: 离心机在 1200 x g 90 分钟在30°c 在培养的板材使用摆动铲斗转子, 并且孵育 3 h 在37°c。</li>
	<li>收集细胞, 转移到一个15毫升管, 并在 450 x g 旋转5分钟 (4 °c)。</li>
	<li>放弃上清, 并用重悬颗粒细胞在6毫升培养基, 放置在 T25 细胞培养瓶, 并在37摄氏度的文化。</li>
	<li>48小时后, 收获细胞和旋转他们在 450 x g 5 分钟 (4 °c)。放弃上清。</li>
	<li>在 gfp 记者的情况下: 将单元格放在 PBS 的500µL 中, 并使用流式细胞仪排序 GFP+单元格。如果嘌呤霉素记者包含向量: 放置细胞在培养基中含有2µg/毫升的嘌呤霉素。</li>
	<li>根据需要展开单元格以进行进一步的分析和实验。 注: (可选) 转基因细胞可以冻结在冷冻容器中, 在-80 摄氏度, 并进一步储存在液氮中。</li>
</ol>4. 32 维/克-脑脊液-R 细胞增殖试验
<ol><li>评估增殖率 0.2 x 106细胞在1毫升培养基和孵化隔夜在37°c。</li>
	<li>第二天计数单元格数, 并将其稀释回浓度为 0.2 x 106细胞/毫升使用培养基。孵化过夜在37摄氏度。使用表 3保存用于日常区域性的单元格浓度和稀释的记录。</li>
	<li>重复步骤 4.2, 因为需要评估扩散的天数。 注: 增殖测定的长度将取决于感兴趣基因的影响。如果有强烈的表型, 8-9 天可能足以观察到一个显著的效果 (请参见图 3A)。然而, 在轻度表型的情况下, 可能需要较长的时间段。</li>
	<li>通过根据表 3中的说明进行扩散曲线来评估细胞生长。</li>
</ol>5. 32 维/克-脑脊液-R 细胞分化试验
<ol><li>清洗 0.2 x 106 32 维/克-脑脊液 R 细胞两次与 RPMI 培养基无细胞因子。 注: 在添加 G 型脑脊液之前, 洗涤步骤是重要的, 因为存在残留 IL-3 可能抑制分化。</li>
	<li>将细胞在1毫升分化培养基中 (含有 100 ng/毫升) 在24井/盘中, 导致细胞浓度为 0.2 x 106细胞/毫升 (天 0)。文化隔夜在37°c。</li>
	<li>如上所示的单元格/mL 数 (步骤 1.2.1)。</li>
	<li>Cytospin 2-5 x 104单元玻璃滑动 (76 毫米 X 26 毫米) 使用离心机与必要的适配器在 20 x g。</li>
	<li>在24井/盘 (天 1) 中, 刷新分化培养基和平板细胞, 浓度为 0.2 x 105细胞/毫升。扔掉旧盘子, 第二天用新盘子的步骤5.6 进行。</li>
	<li>在2到7天, 重复步骤5.3 到5.5。按照步骤4中的说明, 评估在脑脊液中出现的细胞增殖。 注意: (重要) 在分化过程中, 细胞增殖减慢, 因此, 在3到4天的时间内, 在脑脊液的存在, 它是足够的培养细胞在较高浓度。</li>
	<li>根据细胞形态学评估32维/克脑脊液 R 细胞的分化状态。<ol>
<li>固定单元从步骤5.4 在甲醇5分钟的 RT 和保持幻灯片的空气干燥。 注: 从0天到7的幻灯片可以存储在 RT, 并同时固定。同样, 他们也可以保持在 RT 在固定后较长的时间。</li>
		<li>染色细胞使用可能 Grünwald 姬姆萨染色后制造商的协议。</li>
		<li>使用显微镜和放大 X40 可视化染色细胞。</li>
		<li>使用图 1上的说明性图片和表 4中的描述, 量化爆炸、intermediately 分化细胞和成熟粒度的数量。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

实验模型的选择是研究的主要课题之一。虽然主要动物和人类细胞被认为能产生最具生物学意义的数据, 但这些模型可能涉及伦理问题, 往往与昂贵和/或复杂的隔离/培养程序相关。初级细胞数量有限, 很难进行基因调控。此外, 主单元格表示由各种单元格类型组成的异构填充, 可能会使数据解释29复杂化。相反, 细胞线提供了一个几乎无限的生物材料来源, 是成本效益, 并允许绕过?...

造血干细胞和祖细胞为生物体提供了大量成熟的细胞, 包括来自髓系谱系 (中性粒、嗜酸性细胞、嗜碱性细胞和单核) 的干细胞。推动从造血干细胞生产髓细胞的过程被称为 myelopoiesis, 适当地生产成熟的髓细胞以响应不断变化的需求是适当应对压力的有机体的先决条件。条件, 如感染和失血。成熟髓细胞的生产不足可能导致无法消除病原体, 减少凝血和其他危及生命的条件1,2。此外, 髓系发育的改变也可能与血液恶性肿瘤有关, 如急性髓细胞白血病 (AML)3。myelopoiesis 的改变可能是由于各种原因造成的, 例如细胞表面受体的缺陷4、转录因子5、受损信号通路6的改变表达、形成的突变激活癌基因7, 或抑癌基因的失活8。
开发了各种方法来研究髓系发育, 并评估了特定基因改变在这一过程中的作用。用于研究 myelopoiesis 的常用方法涉及原代细胞和转基因小鼠。虽然这些模型允许获取生物相关的数据, 但它们有一定的局限性。使用初级细胞遇到有限的细胞数量和限制的文化周期, 缩小改变基因表达和随后的生物或生化分析的可能性。转基因小鼠成本高昂, 需要合理程度的生物合理性。此外, 使用体内模型也增加了一定程度的复杂性, 从而理解了在给定过程中感兴趣的基因的作用。因此, 需要采取其他办法来规避这些限制。细胞系具有无可争辩的优势: (1) 它们具有无限的增殖能力, 允许产生足够的物质进行生物化学和生物研究, (2) 它们容易受到基因的操控 (击倒, 挖空,过度表达), (3) 成本相对较低, (4) 它们允许某些实验方法所需的生物简化程度。
父母 IL-3 (Interleukin-3) 依赖32D 细胞系成立于1983年由 Greenberger 和同事通过感染的骨髓细胞从 C3H/HeJ 鼠与朋友小鼠白血病病毒9。文献中描述了几个32D 克隆: cl-239、cl-310和 cl-1011。32D cl-3 细胞在 IL-3 中增殖, 并经粒细胞集落刺激因子 (CSF)10治疗中性分化。相反, 32D cl-10 细胞, 而 IL-3 依赖, 最初并没有区别于对 G 脑脊液治疗的反应。在 1995年, Touw 博士 retrovirally 转基因 32D cl-10 细胞与野生类型和突变形式的 g-脑脊液受体 (g 脑脊液 R), 以确定该受体的功能重要区域11。这项研究结果产生的32维/克脑脊液 R 细胞, 这是同样依赖于 IL-3, 但在6至10天后, 替换 IL-3 与 G 脑脊液, 细胞停止增殖和不可逆转地分化成成熟的中性粒细胞。这些特性使 32D cl-3 和32维/克脑脊液 R 细胞简化模型的小鼠中性分化, 可由两个明确的增长和分化因子-IL-3 和 G 脑脊液。在过去的几十年中, 多组使用了32维/克脑脊液 R 细胞来研究特定基因在培养过程中细胞增殖和分化中的作用12,13,14,15,16, 并研究 G CSF 信号17,18。重要的是, 使用该细胞线获得的结果与主要细胞和转基因小鼠的数据相关,16,19,20,21。因此, 我们认为, 32 维/克脑脊液 R 细胞作为一种广泛使用和建立良好的模型, 是研究髓样分化的一个有价值的系统, 可与处理这个问题的其他方法并行使用。
在这里, 详细的协议描述处理32维/克-脑脊液 R 细胞线, 包括扩展, 分化, 并评估这些细胞的增殖和分化。提供了32维/克-脑脊液 R 细胞的基因修饰的详细信息, 可以采用逆转录病毒或慢病毒载体转导, 以及病毒滴定的协议。此外, 还提供了一些表明潜在应用32维/克-脑脊液 R 细胞的典型结果。

<table><tbody><tr><td>RPMI 1640 powder medium</td><td>Merck, Kenilworth, NJ, USA</td><td>T 121-10</td><td>without NaHCO3, with L-glutamine</td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>15028</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>31985-047</td><td>L-Glutamine, Phenol Red</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>PAA Laboratories (GE Healthcare,Chicago, IL, USA)</td><td>MT35011CV</td><td>For differentiation of 32D/G-CSF-R cells</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>10270</td><td>Used for culturing HEK293T, NIH3T3, BOSC23 cells</td></tr><tr><td>Penicillin</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>P3032</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>S9137</td><td>Streptomycin sulfate salt powder</td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>G1914</td><td></td></tr><tr><td>murine IL-3</td><td>PeproTech, Rocky Hill, NJ, USA</td><td>213-13</td><td></td></tr><tr><td>human G-CSF</td><td>PeproTech, Rocky Hill, NJ, USA</td><td>300-23</td><td></td></tr><tr><td>Polyethylenimine</td><td>Polyscience, Warrington, PA, USA</td><td>23966</td><td>Linear, MW 25,000 (PEI 25000)</td></tr><tr><td>Polybrene</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>H9268</td><td></td></tr><tr><td>Trypsin</td><td>VWR Chemicals, Radnor, PA, USA</td><td>0458</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>E5134</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>C0775</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>T6146</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td><td>D2650</td><td></td></tr><tr><td>May-Gr&amp;uuml;nwald Giemsa</td><td>DiaPath, Martinengo, BG, Italy</td><td>10802</td><td></td></tr></tbody></table>

proliferation and differentiation of murine myeloid precursor 32d/g-csf-r cells

对造血干细胞和祖细胞生物学的理解对再生医学和血液病理学的治疗具有重要意义。尽管可以使用体内模型或初级区域性获取最相关的数据, 但造血干细胞和祖细胞的低丰度大大限制了适当技术的研究。因此, 使用细胞线可以充分生产生物材料, 以执行需要大量细胞数的筛查或化验。在这里, 我们提供了一个详细的描述, 读数, 和解释的增殖和分化的检测, 用于调查过程中涉及 myelopoiesis 和中性分化。这些实验采用32维/克-脑脊液-R 细胞因子依赖的小鼠髓细胞系, 具有在 IL-3 存在时增殖的能力, 并能分化为 g 脑脊液。我们为处理32维/克-脑脊液 R 细胞提供了最优化的协议, 并讨论了可能危及所描述的化验结果和预期效果的主要缺陷和缺点。此外, 本文还包含了慢病毒载体和逆转录病毒的生产、滴定和转导32维/克-脑脊液 R 细胞的协议。我们证明, 这些细胞的遗传操作可以用来成功地进行功能和分子研究, 这可以补充取得的结果与原发性造血干细胞和祖细胞或在体内模型。

32维/G-脑脊液 R 细胞的增殖与分化
为评估32维/g-脑脊液 r 细胞在促增殖和诱导分化条件下的增殖, 分别在含有 IL-3 和脑脊液的培养基中培养出32维/克脑脊液 r 细胞。据观察, 在含有培养基的 IL-3 中培养的细胞 (10 ng/毫升) 大约每 24 h (图 2A) 分。在更换 IL-3 时 (100 ng/毫升) 的增殖逐渐减慢并在4天后停止 (<strong clas...

Watch this Scientific Journal Video about Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells at JoVE.com

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

本文介绍了培养小鼠髓质前体32维/G-脑脊液 R 细胞系的详细规程, 并进行了病毒感染, 并进行了增殖和分化测定。该细胞系适合研究髓细胞发育, 以及兴趣基因在髓细胞生长和中性分化中的作用。

小鼠髓质前体 32-脑脊液-R 细胞的增殖和分化

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

proliferation-differentiation-murine-myeloid-precursor-32dg-csf-r

Research

JoVE Journal

Developmental Biology

9.8K 次观看.  Institute of Molecular Genetics of the ASCR. 本文介绍了培养小鼠髓质前体32维/G-脑脊液 R 细胞系的详细规程, 并进行了病毒感染, 并进行了增殖和分化测定。该细胞系适合研究髓细胞发育, 以及兴趣基因在髓细胞生长和中性分化中的作用。

视频: Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

详细的定性C1498小鼠细胞系及其关联白血病小鼠模型协议

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

科研

癌症研究

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

生物学

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

发育生物学

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

人脐带血衍生CD34的泛骨髓分化–造血干细胞和祖细胞

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

免疫与感染

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

小鼠致耐受性树突状细胞的发育及功能表征

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

医学

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Source: Kozicky, L. et al., <a href="https://app.jove.com/v/56689">Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the activation of bone marrow-derived macrophages in response to bacterial lipopolysaccharide and intravenous immunoglobulin.

响应抗体激活小鼠骨髓来源的巨噬细胞

Source: Kozicky, L. et al., Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation. J. Vis. Exp. (2017)
In ...