使用康可藻红素刺激冷冻保存的猪外周单个核细胞进行增殖检测，并结合FCS ExpressTM 7.18软件分析

Marlene Bravo-Parra; Rahul K. Nelli; Lu Yen; Gino Castillo; Juan Carlos Mora-Díaz; Ana F. Castillo-Espinoza; Luis G. Giménez-Lirola

doi:10.21769/BioProtoc.5332

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Peer-reviewed

Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS Express^TM 7.18 Software

使用康可藻红素刺激冷冻保存的猪外周单个核细胞进行增殖检测，并结合FCS ExpressTM 7.18软件分析

MB Marlene Bravo-Parra ^§

RN Rahul K. Nelli

LY Lu Yen

Gino Castillo

JM Juan Carlos Mora-Díaz

AC Ana F. Castillo-Espinoza

LG Luis G. Giménez-Lirola email

(§Technical contact: mbravo@iastate.edu) 发布: 2025年06月05日第15卷第11期 DOI: 10.21769/BioProtoc.5332 浏览次数: 2071

评审: Shaobin ShangAnonymous reviewer(s)

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Abstract

In vitro lymphocyte proliferation assays are essential for assessing immune responses and antiproliferative drug efficacy. Such assays rely on antigen presentation or mitogen stimulation, with performance determined by reagent concentration and incubation time. Although splenocytes are often used, peripheral blood mononuclear cells (PBMCs) offer more accessible and practical sampling. However, a streamlined protocol for porcine PBMCs proliferation with robust batch analysis has been lacking. We therefore developed a detailed workflow for inducing proliferation in cryopreserved porcine PBMCs using 5 μg/mL concanavalin A (ConA). The protocol covers cell isolation, cryopreservation, ConA stimulation, CD4+ T-cell staining, flow cytometry acquisition and gating on an Attune NxT instrument, and batch analysis with FCS Express^TM 7.18. This approach yielded 78.9% viable cells, of which 33.8% were CD4+ lymphocytes. Moreover, 93.9% (n = 216) of cells proliferated, yielding up to nine cell generations. Batch analysis in FCS Express^TM enhanced the accuracy and interpretation of proliferation metrics. This validated protocol provides a reliable framework for generating consistent proliferation data in porcine immunology studies.

Key features

• Optimized proliferation assay for cryopreserved porcine PBMCs: Ideal for immunological studies and biomedical research using swine models.

• Precise acquisition and gating with Attune NxT flow cytometer: Ensures accurate identification and analysis of CD4+ T lymphocytes.

• Robust data analysis using FCS Express^TM 7.18 software: Facilitates reliable interpretation of lymphocyte proliferation and division indexes.

• Efficient 4.5-day protocol: Enables timely and reproducible experiments in clinical and research settings involving porcine immune cells.

Keywords: Proliferation assay (增殖检测)

Porcine peripheral blood mononuclear cells (PBMCs) (猪外周血单个核细胞（PBMCs）)

CD4+ T lymphocytes (CD4⁺ T淋巴细胞)

Concanavalin A (ConA) (康可藻红素（ConA）)

Flow cytometry (流式细胞术)

FCS Express^TM 7.18 software (FCS Express^TM 7.18软件)

Swine models (猪模型)

Graphical overview

Proliferation assay process from cryopreserved porcine peripheral blood mononuclear cells (PBMCs)

Background

Proliferation assays are fundamental for evaluating both the cytotoxic effects of antiproliferative agents [1,2] and antigen-driven immune responses [3]. These assays rely on either antigen presentation or mitogen stimulation, with concanavalin A (ConA) and phytohemagglutinin (PHA), Ca²⁺/Mn²⁺-dependent lectins that bind mannose and glucose residues, serving as indispensable polyclonal stimulants [4,5]. A variety of proliferation readouts have been described: thymidine‐analog incorporation like BrdU [6,7], generational‐tracking dyes such as CFSE and CellTrace [8–10], and detection of proliferation‐associated antigens (Ki67, PCNA) by flow cytometry or western blot [11,12]. Data analysis platforms, including ModFit, FlowJo, and FCS Express^TM, facilitate automated calculation of division and proliferation indexes [13–16].

In swine immunology, proliferation assays underpin vaccine evaluation, disease pathogenesis research, and immune surveillance. However, porcine peripheral blood mononuclear cells (PBMCs) present unique challenges: cryopreservation can reduce viability by 10%–20% and disproportionately impair CD4 T‐cell recovery, while baseline frequencies of CD4⁺ subsets vary widely between animals [17,18]. Published protocols (often developed for fresh splenocytes) lack harmonized freezing/thawing procedures, standardized staining protocols, and data analysis workflows, leading to inter‐laboratory variability.

To overcome these limitations, we developed a fully standardized protocol for inducing proliferation in cryopreserved porcine PBMCs. Cells were stimulated with 5 μg/mL ConA, divisions were tracked via CellTrace^TM Oregon Green dilution, and gating was performed automatically on an Attune NxT flow cytometer. Data were exported in batch mode and analyzed in FCS Express^TM 7.18 using fixed‐parameter proliferation fits with a uniform coefficient of variation, fixed peak ratios, and controlled background noise. This workflow yields highly reproducible division and proliferation indexes across large cohorts, outperforming methods that rely on fresh cells or manual gating and providing a robust foundation for porcine immunology studies.

This protocol is broadly applicable in swine disease research as a reliable positive-control assay for benchmarking proliferation. It facilitates optimization of antigen doses, such as microbial proteins or vaccine candidates, by enabling comparative analysis of proliferative responses across titration series, and supports evaluation of immunomodulatory or antiproliferative compounds under real-world cryopreservation conditions. By employing uniform gating and batch-mode analysis, the method also standardizes comparisons among lymphocyte subsets (e.g., CD4⁺, CD8⁺, γδ T cells), minimizing inter-assay variability. Moreover, its high-throughput format and consistent readouts make it suitable for large-scale vaccine efficacy and challenge studies, where correlating proliferation metrics with clinical outcomes is critical. Incorporating detailed guidelines for sample handling, staining, acquisition, and batch-mode data analysis, this protocol enhances reproducibility and comparability across cohorts and laboratories, thereby advancing swine immunology and vaccine development efforts.

Although ConA is used here as a nonspecific positive control, this workflow can be directly adapted for antigen-specific proliferation assays. By substituting ConA with the antigen or peptide pool of interest (at a concentration determined by preliminary titration) and including unstimulated and ConA-stimulated wells as negative and maximal controls, respectively, one can reliably quantify antigen-driven CD4⁺ T-cell proliferation using the same CellTrace^TM labeling, flow cytometry acquisition, and batch-mode FCS Express^TM 7.18 analysis. In a pilot study, we applied this protocol with influenza virus (IAV) nucleoprotein (NP) at 10 μg/mL in pigs with prior exposure and detected antigen-specific CD4⁺ proliferation, demonstrating the method’s adaptability (data not shown).

Materials and reagents

Biological materials

1. Frozen porcine peripheral blood mononuclear cells PBMCs (5–10 × 10⁶ cells/mL) in freezing media (see Recipes); preserved in liquid nitrogen

Reagents

1. Ficoll-Paque (Cytiva, catalog number: 17-1440-03)

2. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, Gibco^®, catalog number: 14715095)

3. 10× red blood cell (RBC) lysis buffer (Santa Cruz Biotechnology, catalog number: sc-296258)

4. Trypan blue 0.4% (Thermo Fisher Scientific, Gibco^®, catalog number: 15250061)

5. 1× phosphate buffered saline (PBS) (Thermo Fisher Scientific, Gibco^®, catalog number: 10010023)

6. Fetal bovine serum (FBS) (Atlas Biologicals, EqualFetal, catalog number: EF-0500-A); inactivated at 56 °C for 30 min and stored at -20 °C

7. Roswell Park Memorial Institute (RPMI) 1640 buffer, medium without L-glutamine and phenol red-free (Thermo Fisher Scientific, Gibco^®, catalog number: 32404014); stored at 4 °C

8. Penicillin streptomycin: 10,000 U/mL penicillin and 10,000 µg/mL streptomycin (pen-strep) (Thermo Fisher Scientific, Gibco^®, catalog number: 15140122); stored at -20 °C

9. 100× GlutaMAX (Thermo Fisher Scientific, Gibco^®, catalog number: 35050061)

10. 100× minimum essential medium non-essential amino acids (MEM NEAA) (Thermo Fisher Scientific, Gibco^®, catalog number: 11140050); stored at 4 °C

11. 1 M HEPES (Thermo Fisher Scientific, Gibco^®, catalog number: 15630080); stored at 4 °C

12. Cytofix/Cytoperm fixation and permeabilization solution (BD Biosciences, catalog number: BDB554722)

13. LIVE/DEAD Fixable Near-IR Dead Cell Stain kit (Thermo Fisher Scientific, Invitrogen, catalog number: L34976); stored at -20 °C

14. CellTrace^TM Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA, SE), cell-permeant, mixed isomers (Thermo Fisher Scientific, Invitrogen, catalog number: C34555); stored at -20 °C

15. PerCP-Cy5.5 mouse anti-pig CD4a, clone 74-12-4, isotype: mouse BALB/c IgG2b, κ (BD, catalog number: 561474); stored at 4 °C

16. UltraComp eBeads Plus compensation beads, stored at 4 °C (Thermo Fisher Scientific, Invitrogen, catalog number: 01-3333-42)

17. ArC reactive beads (Thermo Fisher Scientific, Invitrogen, catalog number: A10346); stored at 4 °C

18. Dimethyl sulfoxide (DMSO) (Millipore-Sigma, catalog number: D2650)

19. Concanavalin A (Cayman Chemical, catalog number: 14951)

20. Isopropanol (VWR Chemicals BDH, CAS number: 67-63-0)

Solutions

1. HBSS 2% FBS (see Recipes)

2. PBMC medium (see Recipes)

3. 1× RBC lysis buffer (see Recipes)

4. Freezing medium (see Recipes)

5. 0.5% PBS/FBS (see Recipes)

6. FACS buffer (see Recipes)

7. 0.008% trypan blue solution (see Recipes)

8. CellTrace^TM Oregon Green 488 Carboxy-DFFDA, SE stock solution (see Recipes)

9. CellTrace^TM Oregon Green 488 Carboxy-DFFDA, SE working solution (see Recipes)

10. LIVE/DEAD Fixable Near-IR Dead Cell Stain stock solution (see Recipes)

11. LIVE/DEAD Fixable Near-IR Dead Cell Stain working solution (see Recipes)

12. PerCP-Cy^TM 5.5 mouse anti-pig CD4a working solution (see Recipes)

13. ConA stock solution (see Recipes)

14. ConA working solution (see Recipes)

Recipes

1. HBSS 2% FBS

Reagent	Final concentration	Quantity or volume
HBSS	n/a	98%
FBS	n/a	2%

2. PBMC medium

Reagent	Final concentration	Quantity or volume
RPMI 1640	n/a	88%
FBS	n/a	8%
Pen-strep	n/a	1%
MEM NEAA	n/a	1%
HEPES	n/a	1%
GlutaMAX	n/a	1%

Note: Filter the buffer through a 0.2 μm filter and store it at 4 °C. Use within one month.

3. 1× RBC lysis buffer

Reagent	Final concentration	Quantity or volume
10× RBC lysis buffer	10×	10%
Purified Millipore water	n/a	90%

Note: Filter the buffer through a 0.2 μm filter and store it at 4 °C.

4. Freezing medium

Reagent	Final concentration	Quantity or volume
PBMC medium (Recipe 2)	n/a	60%
FBS	n/a	30%
DMSO	n/a	10%

Note: Filter the buffer through a 0.2 μm filter and store it at 4 °C.

5. 0.5% PBS/FBS

Reagent	Final concentration	Quantity or volume
PBMC medium (Recipe 2)	n/a	99.5%
FBS	n/a	0.5%

Note: Filter the buffer through a 0.2 μm filter and store it at 4 °C.

6. FACS buffer

Reagent	Final concentration	Quantity or volume
1× PBS	n/a	99%
FBS	n/a	1%

Note: Filter the buffer through a 0.2 μm filter and store it at 4 °C.

7. 0.008% trypan blue solution

Reagent	Final concentration	Quantity or volume
1× PBS	n/a	9.8 mL
0.4% trypan blue	n/a	0.2 mL
Total	n/a	10 mL

8. CellTrace^TM Oregon Green 488 Carboxy-DFFDA, SE stock solution

Reagent	Final concentration	Quantity or volume
CellTrace-Oregon Green 488 Carboxy-DFFDA	n/a	1 vial
DMSO	n/a	50 μL

Note: Prepare the stock solution before use (1 h) because the DMSO from the kit is frozen, and you need to wait until it thaws. Keep it at room temperature before using it.

9. CellTrace^TM Oregon Green 488 Carboxy-DFFDA, SE working solution

Reagent	Final concentration	Quantity or volume
CellTrace^TM Oregon Green 488 Carboxy-DFFDA, SE stock solution (Recipe 8)	n/a	0.3 μL
1× PBS	n/a	1 mL

Note: Prepare the working solution immediately before use (within 5–10 min). Keep it on ice and protected from light to preserve reagent activity.

10. LIVE/DEAD Fixable Near-IR Dead Cell Stain stock solution

Reagent	Final concentration	Quantity or volume
LIVE/DEAD Fixable Near-IR Dead Cell Stain	n/a	1 vial
DMSO	n/a	50 μL

Note: Prepare the stock solution before use (1 h) because the DMSO from the kit is frozen, and you need to wait until it thaws. Keep it at room temperature and covered from light before using it.

11. LIVE/DEAD Fixable Near-IR Dead Cell Stain working solution

Reagent	Final concentration	Quantity or volume
LIVE/DEAD Fixable Near-IR Dead Cell Stain stock solution (Recipe 10)	n/a	1 μL
1× PBS	n/a	99 μL

Note: Prepare the working solution before use (1 h). Keep it on ice, covered from light.

12. PerCP-Cy^TM 5.5 mouse anti-pig CD4a working solution

Reagent	Final concentration	Quantity or volume
PerCP-Cy^TM 5.5 mouse anti-pig CD4a (200 μg/mL)	2 μg/mL	1 μL
FACS buffer (Recipe 6)	n/a	99 μL

Note: Keep it on ice and covered from light.

13. ConA stock solution

Reagent	Final concentration	Quantity or volume
ConA, lyophilized powder	1 mg/mL	1 mg
1× PBS	n/a	1 mL

Note: Keep it on ice before use.

14. ConA working solution (for 50 wells of 150 μL)

Reagent	Final concentration	Quantity or volume
ConA stock solution (1 mg/mL) (Recipe 13)	5 μg/mL	37.5 μL
PBMC medium (Recipe 2)	n/a	7462.5 μL

Note: Prewarm the ConA working solution before adding it to the cells.

Laboratory supplies

1. 15 mL SepMate tubes (STEMCELL Technologies, catalog number: 85415)

2. 15 mL conical centrifuge tubes NUNC (Thermo Fisher Scientific, Nunc^TM, catalog number: 12-565-269)

3. 10 mL serological pipettes (Thermo Fisher Scientific, Fisherbrand, catalog number: 13-678-11E)

4. 25 mL serological pipettes (Thermo Fisher Scientific, Fisherbrand, catalog number: 13-678-11)

5. Sterile pipette tips 250 μL filtered, non-autoclavable, sterile, DNase- and RNase-free (Biotix, catalog number: M-0250-9NC)

6. Sterile pipette tips 1000 μL, non-autoclavable, sterile, DNase- and RNase-free (Thermo Fisher Scientific, Fisherbrand, catalog number: 02-707-404)

7. DNA LoBind 1.5 mL tubes (Eppendorf, catalog number: 022431021)

8. 500 mL bottles (Thermo Fisher Scientific, Nalgene, catalog number: 02540263)

9. Filter unit 1 L (Thermo Fisher Scientific, Nalgene, catalog number: 0974103)

10. 2 mL cryovials (Corning, catalog number: 430488)

11. Freezing container (Thermo Fisher Scientific, catalog number: 15-350-50)

12. Cell culture microplate, 96 well, PS, U-bottom, clear (Greiner Bio-One, catalog number: 650-180)

13. 40 μm Flowmi cell strainer (SP Bel-Art, catalog number: H13680-0040)

14. Hemocytometer counting chamber (Hausser Scientific, catalog number: 3200)

15. Reagent reservoirs 25 mL (VWR, catalog number: 89094-662)

Equipment

1. Laminar flow hood, Class II Type A2 (Nuaire Biological Safety Cabinets)

2. Incubator, 37 °C, 5% CO₂ (Caron Oasis CO₂)

3. Inverted microscope (Olympus, model: CKX41)

4. Sorvall Legend RT+ centrifuge (Thermo Fisher Scientific, catalog number: 75004377, model: D-37520 Osterode)

5. Attune NxT Gen, Acoustic Focusing Cytometer with Autosampler (Thermo Fisher Scientific, Invitrogen^TM, model: AFC2)

6. Multichannel pipette 30–300 µL (Fisherbrand Elite, catalog number: OU38517)

Software and datasets

1. Attune NxT software v3.2.1 (Thermo Fisher Scientific, Invitrogen^TM)

2. FCS Express^TM 7.18 software, Research Edition (De Novo Software 2023)

3. GraphPad Prism 10 (Dotmatics)

Procedure

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文章信息

稿件历史记录

提交日期: Jan 26, 2025

接收日期: May 6, 2025

在线发布日期: May 26, 2025

出版日期: Jun 5, 2025

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Bravo-Parra, M., Nelli, R. K., Yen, L., Castillo, G., Mora-Díaz, J. C., Castillo-Espinoza, A. F. and Giménez-Lirola, L. G. (2025). Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS Express^TM 7.18 Software. Bio-protocol 15(11): e5332. DOI: 10.21769/BioProtoc.5332.