从全血中分离和冷冻保存高活力的人外周血单核细胞：初学者指南

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.......

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.......

Materials

Name	Company	Catalog Number	Comments
1000 µL Tips	Gilson	F174501	Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
15 mL tube	Biopioneeer	CNT-15	To hold Solutions 2/3 2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
2 mL Cryovials	Globe Scientific	3012	Approximately 40 cryovials needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
20 µL Tips	Gilson	F174201	For 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 Tube	CEM Corporation	50-187-7683	2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection 3 needed per 1 mL of unpooled frozen PBMCs to be defrosted
CD14 Antibody	Biolegend	325621
CD16 Antibody	Biolegend	360723
CD19 Antibody	Biolegend	363007
CD3 Antibody	Biolegend	300405
CD56 Antibody	Biolegend	318303
CD66b Antibody	Biolegend	305103
Cell Strainer	Biopioneeer	DGN258367	1 needed per 1 mL of unpooled frozen PBMCs to be defrosted
CPT Mononuclear Cell Preparation Tube	BD Biosciences	362753	6 tubes needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Cryo Freezer Box	Southern Labware	SB2CC-81	Holds 81 tubes 1 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Cryotube Rack	Fisherbrand	05-669-45	Holds up to 50 cryovials
DMSO	Invitrogen	15575020	Approximately 2.5 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
DNase/RNase-Free Distilled Water	Invitrogen	10977015	Approximately 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 Tubes	BD Biosciences	366643	1 tube needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Flavopiridol	Sigma-Aldrich	F3055-5MG
HLA-DR Antibody	Biolegend	307609
Human Serum Albumin	GeminiBio	800-120	Approximately 25 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection
Human Trustain FcX	Biolegend	422301
Isopropanol	Sigma-Aldrich	W292912-1KG-K	For use in Mr. Frosty
Label Printer	Phomemo	M110-WH
Live-Dead Stain	Biolegend	423105
Mr. Frosty	Thermo Scientific	5100-0001	Holds up to 18 tubes. 2 Mr. Frostys needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Multidispense Pipette	Brandtech	705110
Multidispense Pipette Tips	Brandtech	705744	Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
P1000 Pipette	Gilson	F144059M
P20 Pipette	Gilson	F144056M	For use in cell counting; volume needed is dependent on method of cell counting used.
Printer Labels	Phomemo	PM-M110-3020	Approximately 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)	Invitrogen	AM9260G	Approximately 40 µL needed per 1 mL of frozen PBMCs to be defrosted
RNase-Free PBS (10X)	Invitrogen	AM9625	Approximately 1 mL needed per 1 mL of frozen PBMCs to be defrosted
RNasin	Promega	N2111
RPMI	Corning	10-040-CV	Approximately 80 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection
Transfer Pipettes	Fisherbrand	13-711-7M	Approximately 5 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Tube Holder	Endicott-Seymour	14-781-15	Holds up to 80 CPT/EDTA Tubes