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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

该方案为收集、储存和解冻外周血单核细胞提供了可访问的指南,适用于下游分析和工作流程,如流式细胞术和 RNA 测序。还演示了等离子和血沉棕黄层收集。

Abstract

外周血单核细胞 (PBMC) 通常用于免疫系统及其对疾病和病原体的反应的生物医学研究。该详细方案描述了从全血细胞中分离、冻存和解冻高质量和高活力 PBMC 的设备、用品和步骤,适用于流式细胞术和 RNA 测序等下游应用。还描述了与 PBMC 平行和同时处理全血血浆和血沉棕黄层的方案。该方案易于遵循,利用密度梯度离心分离 PBMC,并附有耗材、设备和制备步骤清单。该方案适用于具有实验室技术经验的个人,并且可以在临床或研究实验室中实施。由没有实验室经验的操作员使用该方案收集的 PBMC 产生高质量的细胞活力和 RNA 测序。

Introduction

该方案展示了一种从全血中分离外周血单核细胞 (PBMC) 的可行方法和工作流程。该方案特别针对新手研究技术人员、学生和临床实验室工作人员,目的是促进 PBMC 的收集和冷冻保存,而无需事先接受过实验室技术培训。

该方案利用离心法按密度分离全血的成分。全血由四种主要成分组成,按密度递减的顺序列出:红细胞/红细胞(占体积的 ~45%)、白细胞(体积的 <1%)、血小板(体积的 <1%)和血浆(体积的 ~55%)1,2,3,4,5,6。白细胞根据其细胞核特性可细分为两类:圆形或多核6。PBMC 定义为具有圆形细胞核的白细胞,由以下细胞类型组成:淋巴细胞(T 细胞、B 细胞、NK 细胞)、树突状细胞和单核细胞6。多核白细胞包括粒细胞,粒细胞由以下细胞类型组成:中性粒细胞、嗜碱性粒细胞和嗜酸....

Protocol

该研究方案已获得 UCSD 和 KUMC 人类保护计划的批准,并符合赫尔辛基宣言。所有个体都提供了参与和采血的知情同意书。

1. PBMC 的处理和冻存

  1. 采血前一天,打印出 表 2 中列出的材料和试剂清单,并相应地准备和标记。
    注:该方案设计用于收集 6 个密度梯度离心管。每管产生大约 1200 万个 PBMC,其中大约 50% 和 20% 分.......

Representative Results

PBMC 收集和冷冻保存后,按照制造商的说明,使用表 4 中列出的试剂通过流式细胞术评估来自 56 个独特样品的解冻 PBMC、单核细胞和淋巴细胞的活力(图 2A-F)。PBMC、单核细胞和淋巴细胞的平均 SD 活力分别为 94 ± 4.0%、98 ± 1.1% 和 93 ± 5.6%(图 2G)。±通过台盼蓝排除法测定活力,平均活力为 88 ± 7.5%,?.......

Discussion

该 PBMC 采集和冷冻保存方案已由接受过和未接受过研究实验室培训的个人成功实施。在我们的应用中,从储存的 PBMC 中纯化的高活力单核细胞的 FACS 和 RNA 测序产生了高质量的序列。

该协议的一个主要优势是它的可访问性。方案中介绍的技术利用预先填充有固体密度梯度介质的试管。因此,可以将全血直接收集到试管中,然后立即处理以分离 PBMC。需.......

Acknowledgements

我们要感谢自愿同意、付出时间和捐献血液样本的患者。我们还感谢堪萨斯大学医学中心的 Patrick Moriarty 博士、Julie-Ann Dutton 和 Mark McClellen 的合作以及在远程站点实施该方案。CY 已获得 NIH 资助 1K08HL150271 的研究支持。

....

Materials

NameCompanyCatalog NumberComments
1000 µL TipsGilsonF174501Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
15 mL tubeBiopioneeerCNT-15To hold Solutions 2/3
2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
2 mL CryovialsGlobe Scientific3012Approximately 40 cryovials needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
20 µL TipsGilsonF174201For use in cell counting; volume needed is dependent on method of cell counting used. 1 tip is needed per 1 mL of unpooled frozen PBMCs to be defrosted
50 mL Conical TubeCEM Corporation50-187-76832 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
3 needed per 1 mL of unpooled frozen PBMCs to be defrosted
CD14 AntibodyBiolegend325621
CD16 AntibodyBiolegend360723
CD19 AntibodyBiolegend363007
CD3 AntibodyBiolegend300405
CD56 AntibodyBiolegend318303
CD66b AntibodyBiolegend305103
Cell StrainerBiopioneeerDGN2583671 needed per 1 mL of unpooled frozen PBMCs to be defrosted
CPT Mononuclear Cell Preparation TubeBD Biosciences3627536 tubes needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Cryo Freezer BoxSouthern LabwareSB2CC-81Holds 81 tubes
1 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Cryotube RackFisherbrand05-669-45Holds up to 50 cryovials
DMSOInvitrogen15575020Approximately 2.5 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
DNase/RNase-Free Distilled WaterInvitrogen10977015Approximately 2 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Approximately 9 mL needed per 1 mL of frozen PBMCs to be defrosted
EDTA TubesBD Biosciences3666431 tube needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
FlavopiridolSigma-AldrichF3055-5MG
HLA-DR AntibodyBiolegend307609
Human Serum AlbuminGeminiBio800-120Approximately 25 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection
Human Trustain FcXBiolegend422301
IsopropanolSigma-AldrichW292912-1KG-KFor use in Mr. Frosty
Label PrinterPhomemoM110-WH
Live-Dead StainBiolegend423105
Mr. FrostyThermo Scientific5100-0001Holds up to 18 tubes.
2 Mr. Frostys needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Multidispense PipetteBrandtech705110
Multidispense Pipette TipsBrandtech705744Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
P1000 PipetteGilsonF144059M
P20 PipetteGilsonF144056MFor use in cell counting; volume needed is dependent on method of cell counting used.
Printer LabelsPhomemoPM-M110-3020Approximately 45 labels needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Approximately 5 labels needed per 1 mL of unpooled frozen PBMCs to be defrosted
RNase-Free EDTA (0.5 M)InvitrogenAM9260GApproximately 40 µL needed per 1 mL of frozen PBMCs to be defrosted
RNase-Free PBS (10X)InvitrogenAM9625Approximately 1 mL needed per 1 mL of frozen PBMCs to be defrosted
RNasinPromegaN2111
RPMICorning10-040-CVApproximately 80 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection
Transfer PipettesFisherbrand13-711-7MApproximately 5 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Tube HolderEndicott-Seymour14-781-15Holds up to 80 CPT/EDTA Tubes

References

  1. Mathew, J., Sankar, P., Varacallo, M. . Physiology, Blood Plasma. , (2018).
  2. Van Oost, B., Van Hien-Hagg, I. H., Timmermans, A. P., Sixma, J. J. The effect of thrombin on the density distribution of blood platelets: Detection of activate....

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