小鼠全眼球消化用于单核吞噬细胞的多参数流式细胞术分析

摘要

本方案提供了一种将完整眼球消化为单细胞悬液的方法，用于多参数流式细胞术分析，以鉴定特定的眼部单核吞噬细胞群体，包括单核细胞、小胶质细胞、巨噬细胞和树突状细胞。

摘要

先天免疫系统在眼部病理生理过程中发挥着重要作用，包括葡萄膜炎、糖尿病视网膜病变以及年龄相关性黄斑变性。先天免疫细胞，特别是单核吞噬细胞，表达重叠的细胞表面标志物，这使得鉴定这些细胞群体具有挑战性。多参数流式细胞术可同时对多种细胞表面标志物进行定量分析，从而区分小鼠眼内的单核细胞、巨噬细胞、小胶质细胞和树突状细胞。本方案描述了完整小鼠眼球的摘除、眼部解剖、消化为单细胞悬液，以及对单细胞悬液进行髓系细胞标志物染色的步骤。此外，我们还解释了如何使用单色对照确定合适的电压，以及如何利用荧光减一对照（fluorescence minus one controls）划定阳性门控范围。多参数流式细胞术的主要局限性在于无法保留组织结构信息。这一局限可通过分别对眼内不同解剖区域进行多参数流式分析，或结合免疫荧光染色加以弥补。然而，免疫荧光技术受限于缺乏定量分析能力，且大多数显微镜可检测的荧光染料数量有限。我们介绍了利用多参数流式细胞术对激光诱导的脉络膜新生血管中单核吞噬细胞进行高通量定量分析的方法。此外，多参数流式细胞术还可用于巨噬细胞亚群的鉴定、细胞命运示踪，以及为转录组学或蛋白质组学研究进行细胞分选。

引言

先天免疫系统包含多种细胞类型，可刺激补体激活和炎症反应。先天免疫细胞包括自然杀伤（NK）细胞、肥大细胞、嗜碱性粒细胞、嗜酸性粒细胞、中性粒细胞以及单核吞噬细胞。单核吞噬细胞由单核细胞、巨噬细胞和树突状细胞组成，已被证实参与多种眼科疾病的病理生理过程，包括葡萄膜炎、糖尿病视网膜病变以及年龄相关性黄斑变性（AMD）¹。在本实验方案中，我们将重点介绍在新生血管性AMD小鼠模型中，利用多参数流式细胞术鉴定单核吞噬细胞的方法²。该方案也可适用于糖尿病视网膜病变和/或葡萄膜炎的小鼠模型，但由于这些疾病具有全身性特征，建议进行更广泛的ocular解剖。

单核吞噬细胞表达重叠的细胞表面标志物。长期存在的组织驻留型巨噬细胞和小胶质细胞起源于卵黄囊来源的红髓系祖细胞³，而循环巨噬细胞和树突状细胞则由骨髓来源的巨噬细胞-树突状细胞祖细胞分化而来⁴。在小鼠中，单核细胞、巨噬细胞和树突状细胞共有的细胞表面标志物包括 CD45、CD11b⁵、F4/80⁶、Cx3cr1⁷，以及细胞内标志物 Iba1⁸。为克服这一难题，通过对多个组织来源的巨噬细胞、单核细胞和树突状细胞进行转录组分析，确定 CD64 为巨噬细胞特异性的细胞表面标志物⁶。在健康眼组织中，虹膜、脉络膜、睫状体和视神经中均已有巨噬细胞的描述³。相比之下，树突状细胞的鉴定更为困难；目前最特异的树突状细胞鉴定方法依赖于使用 Zbtb46-GFP 报告小鼠进行细胞命运图谱分析⁹。除该报告小鼠外，通过 CD11c 和 MHCII 的表达结合 CD64 的缺失，也可识别潜在的树突状细胞^6,10。在正常眼组织中，角膜、结膜、虹膜和脉络膜中均已鉴定出树突状细胞¹¹。小胶质细胞是位于视网膜内的特化巨噬细胞，受血-视网膜屏障保护，并起源于卵黄囊前体细胞¹²。因此，视网膜小胶质细胞可通过其 CD45 表达水平较低¹³以及 Tmem119 高表达（已有适用于流式细胞术的抗体）与单核细胞来源的巨噬细胞相区分¹⁴。然而，在小胶质细胞活化后，CD45 可能上调¹⁵，而 Tmem119 可能下调³，这体现了小胶质细胞生物学的复杂性，在年龄相关性黄斑变性（AMD）及其小鼠模型中可能具有重要意义。最后，单核细胞至少可分为两类亚型：经典型和非经典型。经典型单核细胞表达 CCR2⁺Ly6C^highCX3CR1^low 表型，而非经典型单核细胞则表达 CCR2^-Ly6C^lowCX3CR1^high 标志物⁵。

由于需要对标志物表达水平进行定量分析（即高水平与低/弱表达水平的区分），多参数流式细胞术是区分眼组织及其他组织中单核细胞、巨噬细胞、小胶质细胞和树突状细胞的理想方法。该方法的其他优势包括可鉴定亚群、能够利用荧光激活细胞分选（FACS）对细胞群体进行分选以用于转录组或蛋白质组学分析，以及命运图谱追踪。多参数流式细胞术的主要缺点在于无法保留组织结构信息。这一局限可通过将眼部解剖为不同的眼内亚区（如角膜、结膜、虹膜、晶状体、视网膜以及脉络膜-巩膜复合体）来克服。此外，可辅以免疫荧光成像进行验证，但该方法受限于可检测标志物的数量，且缺乏可靠的定量能力。

全基因组关联研究已将多种补体基因与年龄相关性黄斑变性（AMD）联系起来¹⁶。补体激活可导致过敏毒素的产生、白细胞募集以及继发性炎症反应。在补体受体缺陷型小鼠中，激光损伤导致的单核吞噬细胞募集减少，且激光诱导的脉络膜新生血管（CNV）面积减小¹⁷。类似地，C-C基序趋化因子受体2（CCR2）基因敲除小鼠因组织中单核细胞募集功能缺陷，也表现出单核吞噬细胞募集减少以及激光诱导的CNV面积减小¹⁸。这些数据表明补体系统和单核吞噬细胞与实验性CNV以及可能的新生血管型AMD相关。支持这一关联的证据显示，新生血管型AMD患者外周血单核细胞上的补体受体表达失调^19,20。这些结果表明AMD与单核吞噬细胞之间存在显著关联。

在本研究中，我们将采用实验性激光诱导的脉络膜新生血管（CNV）模型，通过多参数流式细胞术对小鼠眼部的单核吞噬细胞群体进行表征。激光诱导的CNV是新生血管性年龄相关性黄斑变性（AMD）的标准小鼠模型，已用于验证当前一线治疗方案的有效性²¹。本实验方案将描述小鼠眼球的摘除、眼部组织解剖、消化制备单细胞悬液、抗体染色、利用单色对照确定激光电压，以及使用荧光减一（FMO）对照进行设门策略分析。有关激光诱导CNV模型的详细描述，请参见先前发表的研究²²。通过本方案，我们将鉴定小胶质细胞、单核细胞、树突状细胞及巨噬细胞群体。此外，我们将利用MHCII和CD11c进一步定义激光诱导CNV模型中的巨噬细胞亚群。

方案

All procedures were approved by the Northwestern University Institutional Animal Care and Use Committee. C57BL/6 mice were group housed at a barrier facility at the Center for Comparative Medicine at Northwestern University (Chicago, IL). All animals were housed in 12/12 h light/dark cycle with free access to food and water.

1. Collection of ocular tissue

1. Sacrifice 10-12-week-old C57BL/6J mice of either sex using regulated administration of carbon dioxide until mouse is unresponsive and no longer inhaling. Perform cervical dislocation or other approved secondary method to ensure euthanasia.
   NOTE: Transcardial perfusion can be performed to remove circulating leukocytes that are not in the ocular tissues. In this experiment, systemic perfusion does not reduce the number of microglia, monocytes, macrophages, or dendritic cells detected in untreated or laser treated whole eyes. Therefore, the majority of these cell types are located in the ophthalmic tissues and this step is optional.
2. To enucleate eyes, push down near the eye socket while placing a pair of curved forceps gently under the eye as it stands out of the socket. Pinch forceps closed under the eye without squeezing the actual eye. Dislodge eye from the surrounding conjunctiva (please see prior publication)²³.
3. Pull eye laterally until the optic nerve is the only remaining connection to detach the eye. The optic nerve often severs with tension, but a small scissor is sometimes necessary to cut the optic nerve. Place the eye in cold 1x Hank’s Buffered Saline Solution (HBSS) with calcium and magnesium in 1.7 mL microcentrifuge tube.

2. Digestion of ocular tissue

1. Prepare the digestion buffer containing 0.1 mg/mL digestion enzyme and 0.2 mg/mL DNase in cold HBSS. Reconstitute 5 mg digestion enzyme in 2 mL of sterile water initially. Prepare an initial dilution of 10 mg/mL DNase in HBSS. For every 10 mL of the digestion buffer (sufficient for 3 animals) mix 9.4 mL of cold HBSS with 0.4 mL of reconstituted digestion enzyme and 0.2 mL of diluted DNase I.
   NOTE: These concentrations were empirically determined over multiple experiments to provide optimal levels of antibody staining of single cells, while reducing cell death. Collagenase D was tried as an alternative to digestion enzyme with reduced cell viability and decreased CD45⁺ cells. Please see Discussion for more details regarding the iterative process to optimize digestion.
2. Under a dissecting microscope at 10x total magnification, remove the remaining conjunctiva and optic nerve using fine forceps and spring scissors in cold HBSS.
3. Move clean eyes to a dry dissecting dish or small weigh boat. Inject 0.15-0.20 mL/eye of digestion buffer via syringe fitted with 30 G needle into the vitreous cavity after two perforating wounds are made in the eye through the visual axis and 90˚ away from this first wound for digestion buffer egress.
4. Mince whole eyes using spring scissors and fine forceps with all pieces smaller than 0.5 mm x 0.5 mm. Dissociate lens tissue with blunt mechanical disruption via forceps to prevent clogging pipette tips in subsequent steps. For each sample, rinse forceps and scissors each with 0.75 mL of digestion buffer into small dish. Use a new dish for each sample.
5. Transfer contents of the dish to a dissociation tube on ice using P1000 pipette taking care to not lose any material in the pipette transfer. Rinse the dish with additional 1.5 mL of digestion buffer bringing the total volume in dissociation tube to 3.25-3.50 mL.
6. Place the dissociation tube inverted onto electronic dissociator and run cycle “m_brain_03_01”, consisting of 1 min unidirectional rotation at 200 rpm at room temperature, which is a pre-loaded program. Ensure all tissue is at the bottom of the dissociation tube in contact with digestion buffer and the rotor for this and all remaining steps.
7. Place the dissociation tube in shaking incubator for 30 min at 200 rpm at 37 ˚C.
8. Repeat steps 2.6 and 2.7 one additional time. Following second 30 min incubation perform one final mechanical digestion using electronic dissociator as in step 2.6.
9. Stop the reaction by adding 10 mL of cold flow buffer and place tubes on ice.

3. Preparation of cell suspension

1. To create single-cell suspension, pour the halted eye digestion reaction through a 40 μm filter placed on the top of a 50 mL conical centrifuge tube. Using the plunger from a 2.5 mL syringe, push any remaining pieces of undigested eye tissue through the filter.
2. Wash the dissociation tube with 10 mL of Flow buffer and rinse filter before using the same plunger to again pass undigested eye tissue pieces through the filter.
3. Repeat step 3.2 using 5 mL of Flow buffer.
4. Wash the dissociation tube and filter with a final 10 mL of Flow buffer.
   NOTE: Total volume of digestion and flow buffer is about 38-40 mL for each sample.
5. Centrifuge the conical tube at 400 x g for 10 min at 4 ˚C. Decant the supernatant without disturbing the cell pellet.
6. Prepare 1x lysing solution by adding 10x lysing solution to sterile water. Break up the pellet by flicking tube against another tube. To lyse red blood cells, add 1 mL of 1x lysing solution for 30 s with swirling at room temperature (RT).
7. Stop the reaction with 20 mL of Flow buffer.
8. Centrifuge the conical tube at 400 x g for 10 min at 4 ˚C. Decant the supernatant without disturbing the cell pellet.
9. Dissociate the pellet by flicking tube against another tube. Add 5 mL of cold 1x HBSS per tube.
10. Centrifuge tubes at 400 x g for 10 min at 4 ˚C. Aspirate supernatant.

4. Staining of cell suspension for mononuclear phagocytes

1. Prepare 0.5 mL of Live/Dead stain per sample by diluting Live/Dead dye 1:5,000 in cold HBSS.
2. Add Live/Dead stain to each sample using a P1000 pipette making sure to dissociate the pellet completely. Transfer samples to 1.2 mL micro titer tubes.
3. Take a small aliquot (10 μL) to count cells using a hemocytometer or automatic cell counter (see 4.4). Incubate samples with Live/Dead stain for 15 min at room temperature in the dark.
4. Count cells using Trypan blue to determine the number of live cells per sample.
   NOTE: Typically, 2 eyes from a 10-12-week-old C57BL/6J mouse contain less than 2 x 10⁶ living cells.
5. Wash samples by adding 400 μL of cold Flow buffer. Centrifuge tubes in a micro titer tube rack at 400 x g for 10 min at 4 ˚C. Aspirate the supernatant without disturbing the cell pellet.
6. Resuspend cells completely in 500 μL of cold Flow buffer. Centrifuge tubes in a micro titer tube rack at 400 x g for 10 min at 4 ˚C. Aspirate the supernatant without disturbing the cell pellet.
7. Block up to 5 x 10⁶ living cells in 50 µL of F_c block (1:50 dilution in cold Flow buffer of purified rat anti-mouse CD16/CD32). If more than 5 x 10⁶ living cells, increase volumes appropriately.
8. Add F_c block to the pellet making sure to completely dissociate the sample. Incubate for 20 min at 4 ˚C.
9. Add 50 μL of antibody staining solution (see Table 1 for choice and amount of each antibody that is included in the solution) per 5 x 10⁶ living cells directly to cells in F_c block and mix completely. Incubate for 30 min at 4 ˚C.
   NOTE: Antibody quantities are dependent upon the flow cytometer configuration and should be titrated independently for every lab and instrument. See Table 2 for instrument configuration of filters and mirrors. To titrate antibodies, start with manufacturer’s recommendation, and calculate the staining index. Perform a test run of varying antibody concentrations (both above and below the manufacturer’s recommendation) and choose the lowest concentration of antibody where the staining index is maintained. Also, ensure that positive staining that is less bright than the single-color control. Use unstained cells to ensure that negatively stained cells are on scale and have a peak at or less than 1 x 10². Use a fluorescence minus one control to define the positively stained cells.
10. Wash samples by adding 700 μL of cold Flow buffer. Centrifuge tubes in a micro titer tube rack at 400 x g for 10 min at 4 ˚C. Aspirate the supernatant without disturbing the cell pellet.
11. Optionally, after antibody staining, the samples can be fixed with 500 μL of 2% paraformaldehyde in Flow buffer. Fix samples for 15 min at room temperature. Then, perform one wash as described in 4.10. Store fixed samples at 4 ˚C. Count beads should be added on the day of flow cytometry data collection. Fixed samples should be run on flow cytometer in 1-2 days.
    CAUTION: Paraformaldehyde is a corrosive and a health hazard.
12. Wash samples again using 500 μL of cold Flow buffer. Centrifuge tubes in a micro titer tube rack at 400 x g for 10 min at 4 ˚C. Aspirate half of the supernatant without disturbing the cell pellet.
13. Resuspend pellets and transfer to a 96 well, U-bottom assay plate. Centrifuge plate at 400 x g for 5 min at 4 ˚C. To decant the supernatant, turn plate over and in one strong shake dislodge the liquid into a sink basin, without disturbing pellet.
14. In a new 1.2 mL micro titer tube, place 50 μL of well vortexed count beads.
15. Resuspend pellets in 96 well plate in 200 μL of cold Flow buffer. Add cell mixture on the top of beads from prior step. Run on flow cytometer in total volume of 250 μL / micro titer tube.

5. Collection of flow cytometry data

1. Turn on and prepare flow cytometer. Instructions listed here are for a modified four laser cytometer (Table 2).
2. Run a test sample, which includes a small aliquot from each sample, to ensure that all cells are on scale for each detector. Adjust voltages so that all cells are on scale.
3. Run single color controls (SCC) for every fluorophore used in staining cocktail (Table 3). Place micro titer tube into a larger 5 mL polystyrene tube for placement onto the flow cytometer stage.
   NOTE: Root fluorophores like BV421 (Pacific Blue), FITC, PE, Alexa647 (APC), and Alexa700 do not require the same antibody as the cocktail (i.e., CD19 PE for CD64 PE). However, tandem fluorophores like PE-CF594, PE-Cy7, or APC-Cy7 require the same antibody for SCC as cocktail due to potential dissociation of the conjugated fluorophores.
   1. Create the SCC for BV421 by adding 2 drops of compensation beads to a 1.2 mL titer tube along with 1 μL of hamster anti-mouse CD11c BV421. Compensation beads are used because CD11c BV421 is an IgG lambda subtype rather than kappa as described in 5.3.3. Incubate for 15 mins at 4 ˚C. Add 0.2 mL of flow buffer.
   2. Create the SCC for the Live/Dead dye by adding one drop of positive staining beads (green cap) from the amine reactive beads, followed by 1 μL of Live/Dead dye. Incubate for 15 min at room temperature in the dark. Add one drop of the negative beads (white cap) from the amine reactive beads. Add 0.2 mL of flow buffer.
   3. Create the remaining SCCs by adding one drop of positive staining beads (green cap) from the anti-rat and anti-hamster Ig kappa bead set, followed by the antibody quantity listed (Table 3) in a 1.2 mL titer tube. Incubate for 15 min at 4 ˚C. Add one drop of the negative beads (white cap) from the anti-rat and anti-hamster Ig kappa bead set. Add 0.2 mL of flow buffer.
   4. Vortex all bead mixtures completely. Store SCCs at 4 ˚C for up to 3 days.
      NOTE: The SCC for fluorescent reporter mice is unstained cells.
   5. When running SCCs, set voltages such that the SCC sample has a peak greater than any fluorescence for a sample of cells in the same detector channel. In addition, voltages should be set so that any SCC has at least a 0.5 Log difference between the histogram peak in the channel of interest versus any other channel (Figure 1).
4. Run samples on “Low” keeping events/second below 4000. If flow rate cannot be lowered appropriately, dilute samples with additional cold flow buffer. Record the volume added as this will be necessary for the count calculation (see representative results).
5. Collect at least 3 x 10⁶ events per sample. This may be higher than the cell number due to pigment granules and debris counting as events on your cytometer.
6. Record a Fluorescence Minus One (FMO) for each fluorophore other than Live/Dead for proper gating strategy in section 6.4. An FMO is a sample that has been stained with all fluorophores except one.
7. Clean and shut down flow cytometer according to manufacturer or facility instructions.

6. Annotation of flow cytometry data

1. Open a new worksheet using analysis software. Import FCS files from cytometer for all samples and single-color controls.
2. Use SCCs to create a compensation matrix.
3. Apply the compensation matrix to your stained samples and FMOs.
4. Use the FMOs to determine positive staining to draw gates.

结果

图1展示了红激光通道下SCC和细胞的未补偿频率直方图：Alexa647、Alexa700和APC-Cy7。在图1A中，品红色线表示Alexa647的SCC峰值位置，而青色线表示预期的0.5对数差异。请注意，Alexa700和APC-Cy7的峰值位于青色线左侧（即亮度较低）。同时注意，所有细胞信号均位于品红色线左侧。任何位于SCC峰值右侧的细胞信号均未进行补偿。根据荧光染料的化学特性，已知特定通道会发生信号溢出至其他通道的情况（即：紫激光：BV421 -> V500；黄绿激光：PE -> PE-CF594；红激光：Alexa647 -> Alexa700，Alexa700 -> APC-Cy7；交叉激光：PE-Cy7 -> APC-Cy7）。如果上述任一条件未满足，可适当调高某一通道的电压，同时将发生溢出的通道电压调低。参见图1B和图1C中红激光通道的其他示例。一旦确定并记...

讨论

多参数流式细胞术可对复杂组织中的多种细胞类型进行定量分析。本报告中，我们描述了在小鼠眼部激光损伤后，通过流式细胞术检测单核吞噬细胞群体（包括单核细胞、树突状细胞以及巨噬细胞亚群）的方法。Liyanage 等人近期报道了一种类似的设门策略，用于检测中性粒细胞、嗜酸性粒细胞、淋巴细胞、树突状细胞（DC）、巨噬细胞、浸润性巨噬细胞和单核细胞²⁷。我们的设门策略主要区别在于加入了CD64标记。Liyanage 等人将树突状细胞定义为CD4^-CD8^-B220^-CD11b⁺CD11c⁺细胞，巨噬细胞定义为CD4^-CD8^-B220^-CD11b⁺CD11c^-NK1.1^-Ly6G^-细胞。我们则将树突状细胞定义为CD4^-CD8^-B220^-Ly6G^-SiglecF<...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
0.6 ml 微型离心管	Fisher Scientific	05-408-120
1.7 ml 微量离心管	Costar	3620
10 ml 移液管	Fisher Scientific	431031
15 ml 离心管	ThermoFisher	339650
1× HBSS，500 ml	Gibco	14025-092
2.5 ml 注射器	Henke Sass Wolf	7886-1
20% 甲醛溶液	电子显微镜科学公司	15713-S
25 ml 移液管	Fisher Scientific	431032
30 G 针头	Exel	26437
3 ml 鲁尔锁型注射器	Fisher Scientific	14955457
41 x 41 x 8 mm 聚苯乙烯称量皿	Fisher Scientific	08-732-112
50 ml 离心管	Falcon	352070
96孔U型底无盖检测板	Falcon	353910
胺反应性微珠	赛默飞世尔科技	A10628
分析软件	FlowJo	v 10
抗大鼠和抗仓鼠Ig kappa微珠试剂盒	BD 生物科学	552845
C57BL/6J 小鼠	杰克逊实验室	664
细胞计数仪	Invitrogen	AMQAX1000
细胞计数板	Invitrogen	C10283
补偿微珠	ThermoFisher	01-1111-42
计数微珠	赛默飞世尔科技	01-1234-42
弯头镊子	Fisher Scientific	16-100-123
消化酶	Sigma Aldrich	5401020001
解剖显微镜	国家光学与科学仪器公司	DC3-420T
解离管	Miltenyi Biotec	130-096-334
DNase	罗氏	10104159001
电子解离仪	Miltenyi Biotec	130-095-937
FACS Diva 软件	BD 生物科学
Fc阻断剂，大鼠抗小鼠CD16/CD32	BD 生物科学	553142
精细镊子	Integra Miltex	17035X
流动缓冲液	Miltenyi Biotec	130-091-221
流式细胞仪	BD 生物科学
流式管	Falcon	352008
仓鼠抗小鼠 CD11c BV421	BD 生物科学	562782
冰桶	Fisher Scientific	07-210-123
活/死染料	赛默飞世尔科技	65-0866-14
裂解液，10倍浓缩液	BD 生物科学	555899
微量离心管及管架	Fisher Scientific	02-681-380
小鼠抗小鼠CD64 PE	BioLegend	139304
小鼠抗小鼠 NK1.1 PECF594	BD 生物科学	562864
P1000 移液器吸头	德文特科学	P2404
P1000 移液器	Gilson	FA10006M
P2 移液器吸头	Eppendorf	22491806
P2 移液器	Gilson	FA10001M
P20 移液器	Gilson	FA10003M
P200 移液器吸头	德文特科学	P2401
P200 移液器	Gilson	FA10005M
移液器	Fisher Scientific	FB14955202
大鼠抗小鼠 B220 PECF594	BD 生物科学	562313
大鼠抗小鼠 CD11b APC-Cy7	BD 生物科学	557657
大鼠抗小鼠 CD19 AlexaFluor 700	BD 生物科学	557958
大鼠抗小鼠 CD19 PE	BD 生物科学	553786
大鼠抗小鼠 CD4 PECF594	BD 生物科学	562314
大鼠抗小鼠 CD45 FITC	赛默飞世尔科技	11-0451-82
大鼠抗小鼠 CD45 PE-Cy7	BD 生物科学	552848
大鼠抗小鼠 CD8 PECF594	BD 生物科学	562315
大鼠抗小鼠 Ly6C	BD 生物科学	561085
大鼠抗小鼠 Ly6G PECF594	BD 生物科学	562700
大鼠抗小鼠MHC II AlexaFluor 700	BioLegend	107622
大鼠抗小鼠 Siglec F PECF594	BD 生物科学	562757
大鼠抗小鼠 Tim4 AlexaFluor647	BD 生物科学	564178
摇床培养箱	Labnet	311DS
弹簧剪	Fine Science Tools	15024-10
无菌细胞筛，40 mm 尼龙网	Fisher Scientific	22363547
无菌水，500 ml	Gibco	A12873-01
水平转子离心机	Eppendorf	5910 R
0.4% 台盼蓝溶液	Gibco	15250061