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Cytokine/Chemokine Secretion Profiles in Differentiated Mouse Muscle Cells

Qiang Xiao1, Jingyu Sun2, Tina Rose1, Dmitri Samovski2, and Phillip Stahl2
1. R &D Department, Bioscience Division, EMD Millipore, Billerica, MA 2. Department of Cell Biology and Physiology, Washington University in St Louis, MO The assay characteristics of Mouse Cytokine/Chemokine Panels
Panel I (32-plex) Panel II (12-plex) 3.2-10,000
EPO: 16-50,000 Fracktalkine: 32-100,000

Panel III (6-plex) 3.2-10,000
IL-20: 16-50,000 IL-23, IL-27, IL-33: 32-100,000

Introduction
Cytokines and chemokines are a group of small proteins that are secreted primarily from immune cells and play essential roles in inflammation and infectious disease. Over the past decade, a great number of studies have demonstrated that some cytokines/chemokines, such as IL-6, are also secreted from adipocytes (adipokines), hepatocytes (hepatokines), and myocytes (myokines). These non-immune-cell-secreted cytokines and chemokines are involved in the regulation of metabolic homeostasis, as well as the development of metabolic diseases such as diabetes, obesity, and cardiovascular diseases. To further study muscle cell cytokines/chemokine secretion systematically, we collected mouse muscle cell (C2C12) culture medium samples at Day 0, 1, 3, 5 after cell differentiation and measured cytokine/chemokine concentrations in these samples using MILLIPLEX® MAP Mouse Cytokine/Chemokine Panels I (32 analytes), II (12 analytes), and III (6 analytes) in multiplex. Among the 50 cytokines/chemokines measured we found nine cytokines/chemokines that were easily detected in C2C12 culture medium samples. Six of them, including IL-6, Eotaxin, IP-10, VEGF-A, Fractalkine and TARC showed a gradual increase in concentration after cell differentiation. These data demonstrated that MILLIPLEX® MAP Mouse Cytokine/Chemokine panels are useful tools for cytokine/chemokine secretion profiling and indicate that muscle cells may secrete more myokines than have been previously reported.

Results
Mouse C2C12 myoblasts were differentiated with differentiation medium (high-glucose DMEM+ 1% glutamine +1% penicillin/streptomycin and 2% horse serum). Cell shape changes were observed (see cell images below) and cell culture medium samples were collected at Day 0, 1, 3, and 5.
Undifferentiated @ Day 0 Differentiated @ Day 3 Differentiated @ Day 5

Standard curve range (pg/mL) Accuracy (%) Intra-assay CV (%) Inter-assay-CV (%)

3.2-10,000 85-107 1-5 4-12

94-108 1-4 6-14

92-118 4-9 10-20

Fifty cytokines/chemokines were measured in culture medium samples. The analytes showing significant concentration changes (%) during differentiation are shown below.
Eotaxin (pg/mL)
400% 350% 300% 250% 200% 150% 100% 50% 0% 4000% 3500% 3000% 2500% 2000% 1500% 1000% 500% 0% Day 0 Day 1 Day 3 Day 5

IL-6 (pg/mL)

Exp#1 Exp#2

Exp#1 Exp#2

C2C12 culture medium samples (25 µL/well) were analyzed for cytokine /chemokine concentrations using MILLIPLEX® MAP Mouse Cytokine/Chemokine panels. These multiplex assays are specific, sensitive, and reproducible. The standard curves are shown below.
Mouse Cytokine/Chemokine Panel I 32-plex Cat#MCYTOMAG-70K, Standard Curves (part I) Mouse Cytokine/Chemokine Panel I 32-plex Cat#MCYTOMAG-70K , Standard Curves (part II)

Day 0 Day 1 Day 3 Day 5

IP-10 (pg/mL)
700% 600% 500% 400% 300% 200% 100% 0% Day 0 Day 1 Day 3 Day 5 Exp#1 Exp#2 450% 400% 350% 300% 250% 200% 150% 100% 50% 0%

VEGF-A (pg/mL)

Methods
C2C12 myoblasts were cultured in growth medium (GM): Dulbecco’s modified Eagle’s medium (DMEM; high glucose) containing 1% glutamine, 1 % penicillin/streptomycin and supplemented with 10 % fetal bovine serum. When the cells reached to 70%-80% confluency in a 10 cm plate, cells were seeded in a 6-well dish. After the cells were approximately 90% confluent in a 6-well dish, the growth medium was switched to a differentiated medium (high-glucose DMEM+ 1% glutamine +1% penicillin/streptomycin and 2% horse serum) before washing the cells with PBS twice. The cells were cultured in differentiated medium for 3-4 days (depending on the differentiated condition of the cells, and samples were collected at Day 0, 1, 3, and 5. MILLIPLEX® technology.
MAP

Exp#1 Exp#2

MFIs

MFIs

Day 0 Day 1 Day 3 Day 5

Fractalkine (pg/mL)
400% 350% 300% 250% 200% 150% Exp#1 Exp#2 200% 150% 100% 50% Day 0 Day 1 Day 3 Day 5 0% 300% 250%

TARC (pg/mL)

Mouse Cytokine/Chemokine Panels using Luminex® xMAP®

Exp#1 Exp#2

Standards (pg/mL)
Mouse Cytokine/Chemokine Panel II 12-plex Cat#MCYP2MAG-73K, Standard Curves

Standards (pg/mL)
Mouse Cytokine/Chemokine Panel III 6-plex Cat#MCYP3MAG-74K, Standard Curves

100% 50% 0%

Day 0 Day 1 Day 3 Day 5

Summary
Mouse C2C12 culture samples were measured using MILLIPLEX® MAP Mouse Cytokine/Chemokine Magnetic Bead Panels (Panel I, 32 analytes, Panel II: 12 analytes, and panel III, 6 analytes). Assays were performed according to the respective protocols. In general, the 96-well assay plate was washed with 200 μL assay buffer per well. To each well was added 25 μL standard/control or buffer, 25 μL culture media or samples, and 25 μL capture Ab conjugated beads. Plates were incubated overnight with shaking at 4°C. The assay plate was washed with wash buffer. 25 μL detection antibody cocktail was added to each well and incubated 1 h at room temperature (RT). After adding 25 μL streptavidin-phycoerythrin (SAPE) to each well, the plate was incubated at RT for 30 min. The assay plate was then washed twice with wash buffer and beads resuspended in 150 μL sheath fluid. All plates were analyzed using the Luminex 200™ instrument.

MFIs

MFIs

We have used MILLIPLEX® MAP Mouse Cytokine/Chemokine Panels I (32 analytes), II (12 analytes), and III (6 analytes) multiplex assays to study muscle cell cytokines/chemokine secretion systematically. Among the 50 cytokines/chemokines tested, we found 9 cytokines/chemokines that were easily detectable in C2C12 culture medium samples, and IL-6, Eotaxin, IP-10, VEGF-A, Fractalkine and TARC showed a gradual increase in concentration after cell differentiation. These data demonstrated that MILLIPLEX® MAP Mouse Cytokine/Chemokine Magnetic Bead Panels are useful tools for cytokine/chemokine secretion profiling, and suggested that muscle cells may secrete more myokines than have been previously reported.

Corresponding Authors: xiao.qiang@emdmillipore.com

Standards (pg/mL)
EMD Millipore and the M logo are trademarks and MILLIPLEX is a registered trademark of Merck KGaA, Darmstadt, Germany. © 2013 EMD Millipore Corporation. All rights reserved. Lit. No. PS5773EN00

Standards (pg/mL)

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