利用重组人半乳糖凝集素体外激活Jurkat E6-1细胞的方法

Ruei-De Yan; Ling Mao; Yi-Jing Chen; Chien-Hui Lo; Yi-Chang Liu

doi:10.21769/BioProtoc.5772

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Peer-reviewed

Protocol for In Vitro Activation of Jurkat E6-1 Cells Using Recombinant Human Galectin

利用重组人半乳糖凝集素体外激活Jurkat E6-1细胞的方法

RY Ruei-De Yan

LM Ling Mao

YC Yi-Jing Chen

CL Chien-Hui Lo email

YL Yi-Chang Liu email

发布: 2026年08月05日第16卷第15期 DOI: 10.21769/BioProtoc.5772 浏览次数: 141

评审: Luis Alberto Sánchez VargasTrupti PatilMartin V Kolev

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Abstract

Surface receptor engagement governs T-cell activation. Since these surface receptors undergo extensive glycosylation, lectin-mediated crosslinking of these glycosylated surface receptors has the potential to modulate signaling. Here, we systematically evaluate the abilities of recombinant human galectins in triggering immune responses. We describe how to apply the human galectins to modulate Jurkat E6-1 cell activation by measuring the expression level of cellular surface CD69 and the mRNA of IL-2. To validate the protocol, we confirmed that galectin-3 and galectin-8 variants 1 and 2 reproducibly induce CD69 and IL-2 expression on Jurkat E6-1 cells. Our approach offers a galectin-based toolset to study how glycosylation modulates human adaptive immunity.

Key features

• Systematic screening of the recombinant human galectin 1, 3, 7, 8 (both of variant 1 and variant 2) for Jurkat E6-1 cell modulatory activity.

• Mechanism-based approach utilizing glycan crosslinking to induce receptor engagement.

• Standardized workflow to evaluate extracellular galectin-driven immune activation.

Keywords: T-cell activation (T细胞活化)

Immunology (免疫学)

Glycobiology (糖生物学)

Recombinant human lectins (重组人凝集素)

Galectins (半乳糖凝集素)

Glycan-binding proteins (糖链结合蛋白)

CD69 (CD69)

IL-2 (IL-2)

Graphical overview

Background

The assembly of an immunological synapse drives T-cell activation [1–3]. Notably, key surface receptors governing T-cell activation (such as the TCR and co-stimulatory molecules) undergo extensive glycosylation. Extracellular lectins can bind to these glycans, inducing receptor crosslinking and subsequent downstream signaling cascades. This glycosylation presents an opportunity to modulate immune responses via glycan-binding proteins [3]. Here, we describe a protocol to achieve T-cell activation by using recombinant human galectins [1]. This protocol describes the specific steps to reconstitute the recombinant human galectin from a lyophilized form. Next, we describe steps for achieving T-cell activation by utilizing recombinant human galectins in a dose-dependent manner. Finally, we provide instructions on determining the IL-2 and CD69 relative expression levels via qPCR and flow cytometry to evaluate the degree of cell activation.

This protocol introduces a comprehensive protocol for in vitro T-cell activation by utilizing recombinant human galectins [1,2]. Unlike standard chemical stimulants such as phorbol 12-myristate 13-acetate/Ionomycin, this protocol utilizes recombinant human galectins to trigger T-cell activation. We systematically evaluate the human galectin family for its capacity to modulate T-cell responses. Our findings identify galectin-3 and galectin-8 as potent triggers of T-cell activation. Using CD69 expression and IL-2 production as indicators, we establish a robust platform for further research in human glycoimmunology.

Materials and reagents

Biological materials

1. Jurkat E6-1 (BCRC, catalog number: 60424)

Reagents

1. PE mouse IgG1, κ isotype Ctrl antibody (anti-mouse IgG1) (BioLegend, catalog number: 400114)

2. APC anti-human CD69 antibody (anti-CD69) (BioLegend, catalog number: 310910)

3. GMP Ultra-LEAF^TM purified anti-human CD3 SF antibody (BioLegend, catalog number: 317353)

4. CD28 monoclonal antibody (CD28.6) (Invitrogen, catalog number: 16-0288-85)

5. Recombinant human galectin 3, His Tag (GlycoGenetics, Inc., catalog numbers: PL02-025, PL02-050, PL02-100)

6. Recombinant human galectin 8, variant 1, His Tag (317 a.a.) (GlycoGenetics, Inc., catalog numbers: PL03-025, PL03-050, PL03-100)

7. Recombinant human galectin 8, variant 2, His Tag (359 a.a.) (GlycoGenetics, Inc., catalog numbers: PL04-025, PL04-050, PL04-100)

8. Recombinant human galectin 1, His Tag (GlycoGenetics, Inc., catalog numbers: PL01-025, PL01-050, PL01-100)

9. Recombinant human galectin 7, His Tag (GlycoGenetics, Inc., catalog numbers: PL05-025, PL05-050, PL05-100)

10. Primer IL-2 forward (IDT, AGAACTCAAACCTCTGGAGGAAG)

11. Primer IL-2 reverse (IDT, GCTGTCTCATCAGCATATTCACAC)

12. Primer GAPDH forward (IDT, GACGCTGGGGCTGGCATTG)

13. Primer GAPDH reverse (IDT, GCTGGTGGTCCAGGGGTC)

14. Phosphate-buffered saline, pH 7.4 (PBS) (Gibco, catalog number: 10010049)

15. Fetal bovine serum (FBS) (Corning, catalog number: 35-010-CV)

16. HyClone^TM penicillin-streptomycin 100× solution (Pen/Strep) (Cytiva, catalog number: SV30010)

17. RPMI-1640 (Gibco, catalog number: 11875119)

18. Sodium pyruvate (Gibco, catalog number: 11360070)

19. Sodium bicarbonate 7.5% solution (Gibco, catalog number: 25080094)

20. HEPES (Gibco, catalog number: 15630080)

21. Glucose solution, 200 g/L (Gibco, catalog number: A2494001)

22. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148-500G)

23. Zymo Quick-RNA Miniprep kit, 200 preps (Zymo Research, catalog number: R1055)

24. HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, catalog number: R323-01)

25. FastSYBR mixture (High ROX), 5 mL (CWBIO, catalog number: CW2622M)

26. Trypan blue solution, 0.4% (Gibco™, catalog number: 15250061)

Solutions

1. RPMI-1640 complete medium (see Recipes)

2. Jurkat E6-1 cell culture medium (see Recipes)

Recipes

1. RPMI-1640 complete medium

Reagent	Final concentration	Quantity or volume
RPMI-1640	n/a	500 mL
FBS	10%	50 mL
Pen/Strep	1×	5 mL
Total	n/a	555 mL

Note: We recommend storing the prepared complete medium at 4 °C for up to 3 months.

2. Jurkat E6-1 cell culture medium

Reagent	Final concentration	Quantity or volume
RPMI-1640 complete medium	n/a	555 mL
Sodium pyruvate	1 mM	5.92 mL
Sodium bicarbonate	1.5 g/L	11.84 mL
HEPES	10 mM	5.92 mL
Glucose solution, 200 g/L	4.5 g/L	13.32 mL
Total	n/a	592 mL

Note: We recommend storing the prepared complete medium at 4 °C for up to 3 months.

Laboratory supplies

1. Thermo MicroAmp Optical 8-Cap Strips (Thermo Fisher Scientific, catalog number: 4323032)

2. Thermo MicroAmp Fast 8-Tube Strip, 0.1 mL (Thermo Fisher Scientific, catalog number: 4358293)

3. Nunc 96-Well, polystyrene, conical bottom, sterile, without lid (Nunc, catalog number: 249662)

Equipment

1. CytoFLEX S (Beckman Coulter, model number: B75442)

2. StepOne Plus^TM Real-Time PCR System (Thermo Fisher Scientific, catalog number: 4376592)

3. PSU-2T Mini shaker (Biosan, catalog number: BS-010155-AAK)

Software and datasets

1. FlowJo (BD Biosciences, v.10.8.1)

2. StepOne Software (Thermo Fisher Scientific, v.2.3)

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: May 17, 2026

接收日期: Jun 29, 2026

在线发布日期: Jul 10, 2026

出版日期: Aug 5, 2026

版权信息

如何引用

Yan, R., Mao, L., Chen, Y., Lo, C. and Liu, Y. (2026). Protocol for In Vitro Activation of Jurkat E6-1 Cells Using Recombinant Human Galectin. Bio-protocol 16(15): e5772. DOI: 10.21769/BioProtoc.5772.