A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

Summary

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

Abstract

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

Introduction

Resting B cells could be transformed by the Epstein-Barr virus (EBV) in vitro into actively proliferating B lymphoblastoid cell lines (B-LCLs). B-LCLs are similar to germ cells in differentiation and development, and the somatic mutation rate of B-LCLs was only 0.3%¹, with negligible genetic and phenotypic alteration. Therefore, B-LCLs, as surrogates for peripheral blood mononuclear cells (PBMCs), have substantially accelerated the progress of genomics, transcriptomics and proteomics study of EBV associated diseases^2,3. Moreover, B-LCLs also have important application in screening monoclonal antibodies^4,5.

Protocol

This study was approved by the Ethics Committee of the Institute of Genetics and Developmental Biology, and the protocol follows the institutional guidelines for human welfare.

1. Preparation of EB Virus

1. On day 1, thaw B95-8 cells and culture in 6 mL complete RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 µg/mL streptomycin, 100 U/mL penicillin) in a T25 culture flask. Place the culture flask in 5% CO₂ incubator at 37 °C.
2. On day 2, observe the cell morphology under a microscope. Cells are in good condition when they are bright and round. Add 10 mL complete RPMI 1640 m....

Results

During the process of transformation, the morphological changes of cells are visualized by light microscopy (Figure 2). Small clusters of cells were clearly visible with the density gradient separation method after 7 days of infection, while only a thick layer of cell fragments are visible from the red blood cell lysis method. However, lymphoblastoid cell clusters are visible under the thick layer of cell fragments 30 days post infection. With more frequent medium change,.......

Discussion

We report the development of a novel method for immortalizing human B cells from whole blood by lysing red blood cells, and its cell viability of established B-LCLs was evaluated by the MTT assay. The results showed that the cell viability of B-LCLs established by the red blood cell lysis method is much higher than that of the density gradient separation method. The major advantage of the red blood cell lysis method is that it is simple and requires a small volume (as little as 0.5 mL) of blood for establishing a permane.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Centrifuge	Techcomp	CT6T	Centrifugation
Automated Cell Counter	Countstar	IC 1000	For cell counting
Epoch Microplate Spectrophotometer	BioTek Instruments	SN263839	For measuring the absorbance
96 well cell culture cluster	Corning Incorporated	3599	Polystyrene plates
24 well cell culture cluster	Corning Incorporated	3524	Polystyrene plates
25 cm2 cell culture flask	Nest	707001	Polystyrene
FBS	Gibco	10270	Component of B-LCLs medium
RPMI Medium 1640	Gibco	31800-022	For B-LCLs medium
L-Glutamine	Amresco	0374	Component of B-LCLs medium
100x streptomycin penicillin solution BioRoYeeBRY-2309	BioRoYee	BRY-2309	Component of B-LCLs medium
Ficoll paque plus	GE  Healthcare	17-1440-03	For in vitro isolation of lymphocyte
PHA-M	Sigma	L8902	For stimulating lymphocyte proliferation
Cyclosporin A	Cayman  Chemical	12088	For inhibiting the cytotoxicity effect of T cells
Red cell lysis buffer	Tiangen	RT122-01	For lysing red blood cell
MTT	Amresco	0793	For the detection of cell viability
DMSO	Sigma	D2650	For freezing cells