Journal: Neuroscience Bulletin

Article Title: Comprehensive Proteomic Profiling of Patients’ Tears Identifies Potential Biomarkers for the Traumatic Vegetative State

doi: 10.1007/s12264-018-0259-x

Figure Lengend Snippet: Gene Ontology analysis revealed 21 proteins involved in response to wounding.

Article Snippet: ELISA and Receiver Operating Characteristic (ROC) Curve Analysis In the verification stage, the levels of 7 promising tear proteins [cystatin B (CTSB), protease, serine 1 (PRSS1), S100 calcium-binding protein A7 (S100A7), glutathione S-transferase P (GSTP1), complement factor H (CFH), kininogen 1 (KNG1), and alpha-1-acid glycoprotein 1 (ORM1)] were measured using the ELISA kits for human CSTB, human PRSS1, human CFH, human KNG1, and human ORM1 (Boster Biological Technology, Wuhan, China) and for human S100A7 and human GSTP1 (from Cloud-clone Corp., Houston, TX).

Techniques:

Journal: Neuroscience Bulletin

Article Title: Comprehensive Proteomic Profiling of Patients’ Tears Identifies Potential Biomarkers for the Traumatic Vegetative State

doi: 10.1007/s12264-018-0259-x

Figure Lengend Snippet: Levels of representative proteins in tears from healthy controls and traumatic vegetative state patients. A List of 7 selected differentially-expressed proteins. Levels of CTSB (B), PRSS1 (C), S100A7 (D), GSTP1 (E), CFH (F), KNG1 (G), and ORM1 (H).

Article Snippet: ELISA and Receiver Operating Characteristic (ROC) Curve Analysis In the verification stage, the levels of 7 promising tear proteins [cystatin B (CTSB), protease, serine 1 (PRSS1), S100 calcium-binding protein A7 (S100A7), glutathione S-transferase P (GSTP1), complement factor H (CFH), kininogen 1 (KNG1), and alpha-1-acid glycoprotein 1 (ORM1)] were measured using the ELISA kits for human CSTB, human PRSS1, human CFH, human KNG1, and human ORM1 (Boster Biological Technology, Wuhan, China) and for human S100A7 and human GSTP1 (from Cloud-clone Corp., Houston, TX).

Techniques:

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Proteomic analysis of the synovial fluid (SF) samples from patients with different stages of temporomandibular joint osteoarthritis (TMJOA). A) Flow diagram for patient inclusion and grouping. B) Photograph showing joint fluid collection during arthroscopic surgery. C) Flow diagram for the proteomic analysis of SF samples. D) Principal component analysis of SF samples. E) Top 10 genes that showed an upward trend and top 10 genes that showed a downward trend in the three groups. F) Orosomucoid 1 (ORM1) expression in patients with different grades of TMJOA, including Mild (n = 21), Moderate (n = 27), and Severe (n = 15) groups. Data is expressed as mean ± SD and analyzed using one‐way analysis of variance (ANOVA) followed by Tukey's post‐hoc test. G) ORM1 expression in patients with pain (n = 30) or without pain (n = 26). Data is expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed). H) ORM1 expression in patients with different degrees of maximum interincisal opening (MIO). Data in MIO ≤ 25 mm (n = 10), MIO 26–35 mm (n = 20), and MIO ≥ 36 mm (n = 29) groups are expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test. I) ORM1 expression in patients with sleep bruxism (n = 26) or without sleep bruxism (n = 23). Data is expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed). J) ORM1 concentration in SF samples detected by ELISA assay in Mild (n = 5), Moderate (n = 5), and Severe (n = 5) groups. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test. FOT, fraction of the total; * p < 0.05.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Expressing, Two Tailed Test, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Histopathological and microcomputed tomography (micro‐CT) analysis showing the effect of ORM1 on maintaining cartilage homeostasis in rats with TMJOA. A) Immunofluorescence analysis of ORM1 in the condylar cartilage of rats with or without TMJOA. Data are expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed), n = 3, * p < 0.05. B) Subcellular localization of exogenous ORM1 conjugated with fluorescein isothiocyanate (FITC). C) Hematoxylin–eosin and Safranin O‐fast green staining of the condylar cartilage of rats in various treatment groups. D) Thickness of the condylar cartilage of rats in various treatment groups based on the Hematoxylin–eosin staining. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with the UAC+NS group. E) Osteoarthritis Research Society International (OARSI) scores of the condylar cartilage of rats in various treatment groups based on the Safranin O‐fast green staining. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with the UAC+NS group. F) Micro‐CT images of condylar tissue samples from various treatment groups. Comparison of percent bone volume (BV/TV) (G), bone surface/volume ratio (BS/BV) (H), trabecular number (Tb.N) (I), trabecular separation (Tb.Sp) (J), and bone mineral density (BMD) K) among various treatment groups. NS, normal saline; UAC, unilateral anterior crossbite. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with the UAC+NS group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Tomography, Micro-CT, Immunofluorescence, Two Tailed Test, Staining, Comparison, Saline

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Bulk RNA sequencing in C28/I2 cells treated with ORM1, and the inhibitory effect of ORM1 on MMP13 and MMP3. A) Administration on C28/I2 cells and the principal component analysis; B) Volcano map showing the differentially expressed genes (DEGs) of interest; C) Top 20 DEGs rescued after ORM1 treatment; D) Expression change of genes related to osteoarthritis in various treatment groups; E) The transcripts per million (TPM) of MMP13, MMP3, MMP12, and MMP9 in the three groups. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, # p < 0.05 compared with the NC group, * p < 0.05. F) GO analysis of DEGs that were upregulated after IL‐1β treatment and downregulated after ORM1 treatment. G) mRNA expression level of MMP13 and MMP3 in both natural and inflammation environments in C28/I2 and SW1353 cells after treatment with ORM1 protein. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, # p < 0.05 compared with the NC group, * p < 0.05. H) Western blot showing the protein level of MMP13 and MMP3 in both natural and inflammatory environments in C28/I2 and SW1353 cells after treatment with ORM1 protein. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, # p < 0.05 compared with the natural control (NC) group, * p < 0.05. I, J) Immunofluorescence analysis showing the effect of ORM1 on MMP13 and MMP3 in vitro. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, # p < 0.05 compared with the NC group, * p < 0.05. K) Immunohistochemical staining of MMP13 and MMP3 in the condylar cartilage of rats among various treatment groups. L) Comparison of MMP13‐ and MMP3‐ positive cells in rats among various treatment groups. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with UAC+NS group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: RNA Sequencing, Expressing, Western Blot, Control, Immunofluorescence, In Vitro, Immunohistochemical staining, Staining, Comparison

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Identification of the interaction between ORM1 and vimentin (VIM). Co‐immunoprecipitation (CoIP) was used to detect binding between ORM1 and VIM in HEK‐293T (A) and C28/I2 cells (B). An immunocolocalization assay was used to examine the colocalization of VIM and exogenous ORM1 (C) or endogenous ORM1 (D) in C28/I2 cells. Data are expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed), n = 3, * p < 0.05. Immunocolocalization (E) and quantification (F) of ORM1 and VIM in the condylar cartilage of rats in the three treatment groups, with the yellow lines showing the articular surface and the interface between bone and cartilage. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05. Western blot (G) and immunofluorescence (H) showing the protein level of VIM in both natural and inflammation environments in C28/I2 and SW1353 cells after treatment with ORM1 protein. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the NC group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Immunoprecipitation, Binding Assay, Two Tailed Test, Western Blot, Immunofluorescence

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Inhibitory effect of ORM1 on VIM/MAPK/MMP signaling pathway. A) Effect of ORM1 treatment on the phosphorylation of ERK, JNK, and p38 in C28/I2 and SW1353 cells was detected using western blot. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the NC group. Expression of MMP13 and MMP3 (B), and the phosphorylation of extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (p38) (C) after overexpression of VIM with or without ORM1 treatment in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the Myc group. Expression of MMP13 and MMP3 (D), and the phosphorylation of ERK, JNK, and p38 (E) after knockdown of VIM in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed), n = 3, * p < 0.05, # p < 0.05 compared with the NC siRNA group. F) Expression of MMP13 and MMP3 after overexpression of VIM with or without inhibition of ERK, JNK, and p38 in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05. G) The phosphorylation levels of ERK, JNK, and p38 in condylar cartilage of rats were detected by immunofluorescence, with the yellow lines showing the articular surface and the interface between bone and cartilage. H) Quantification of the phosphorylation levels of ERK, JNK, and p38 in condylar cartilage of rats based on the immunofluorescence. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with the UAC+NS group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Phospho-proteomics, Western Blot, Expressing, Over Expression, Knockdown, Two Tailed Test, Inhibition, Immunofluorescence

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: The role of ORM1‐VIM binding on the activities of MAPK pathway and the expression of MMPs. A) Immunocolocalization of ORM1 truncations and VIM in C28/I2 cells. B) CoIP of ORM1 truncations and VIM in C28/I2 cells. C) Expression of MMP13 and MMP3 after overexpression of VIM without or with the ORM1 truncations in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the Myc+Flag group. D) Phosphorylation of ERK, JNK, and p38 after overexpression of VIM without or with the ORM1 truncations in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the Myc+Flag group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Binding Assay, Expressing, Over Expression, Phospho-proteomics

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Schematic diagram of the role of ORM1 in maintaining cartilage homeostasis via suppressing VIM/MAPK/MMP signaling.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques:

Journal: Genome Biology

Article Title: Extensive rewiring of epithelial-stromal co-expression networks in breast cancer

doi: 10.1186/s13059-015-0675-4

Figure Lengend Snippet: Genes predicted to be involved in epithelial-stromal self-loops show coordinate epithelial and stromal protein expression by immunohistochemistry. a Scatterplots of mRNA expression of MX1 , ORM1 , SERPINA1 , and TOM1L1 in epithelium and stroma of 82 invasive breast carcinomas from LCM-derived gene expression data. The epithelial and stromal expression of each gene is positively correlated in cancer. b Protein expression of MX1, ORM1, SERPINA1, and TOM1L1 is concurrently seen in cancer epithelium and stromal spindle cells in images from the Human Protein Atlas. Stronger protein expression is often seen at the periphery of tumor nests and at the tumor-stroma interface for each protein marker ( black bar 100 um, black arrow cancer epithelium; open arrow stromal cells)

Article Snippet: The epithelial and stromal expression of each gene is positively correlated in cancer. b Protein expression of MX1, ORM1, SERPINA1, and TOM1L1 is concurrently seen in cancer epithelium and stromal spindle cells in images from the Human Protein Atlas.

Techniques: Expressing, Immunohistochemistry, Derivative Assay, Gene Expression, Marker

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Orm1 is induced in humans and mics after PH. (a)–(c) Human data. (a) Postoperative serum Orm1 levels were examined in 10 patients who had undergone liver resection for HCC. (b) Correlation between changes in serum Orm1 at 24 h post-liver resection with the rates of liver resection (the resection volume counts per total liver volume counts). POD, postoperative days. (c) Representative photographs of IHC staining of Orm1 in commercially obtained liver sections of HCC and normal adjacent tissues in HCC patients. As a negative control (Ctl), human Orm1 antibody that had been incubated with a 5-fold (by weight) excess of blocking peptide. (d)–(g) Mouse data. (d) Time course of Orm1 gene expression in whole-liver tissues after PH (n = 3). (e) Orm1 transcript levels in whole-liver tissues and (f) serum protein levels at 48 h after PH (n = 4). (g) IHC staining of Orm1 in mouse liver tissues at 48 h after PH. P -value was assessed using Student's t -test or Mann-Whitney U test. * P < 0.05 indicates statistical significance. Scale bars, 100 μm.

Article Snippet: For gain-of-function analysis, 24 h after siRNA transfection, FLC4 cells were treated with recombinant human Orm1 (CLPRO889, Cedarlane, Burlington, NC, USA) in DMEM containing 2% FBS for 24 h before cell viability determination.

Techniques: Immunohistochemistry, Negative Control, Incubation, Blocking Assay, Gene Expression, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Transcriptional profiling of LSECs and HPCs during liver regeneration. (a) Schematic overview of transcriptome experimental procedures (n = 3). (b) Hierarchical clustering with Ward's method of 16,499 genes measured by CAGE technology in LSECs and HPCs. The mean average from three biological replicates at each time point was shown. Low expression tags were deleted and the tags that were expressed at least at one time point in either LSECs or HPCs were kept with the expression cutoff at 1 tags per million (TPM). PCA score plots of differentially expressed genes in the process of liver regeneration for (c) LSECs and (d) HPCs. Numbers of significantly differentially expressed genes compared with the sham control at each time point in (e) LSECs and (f) HPCs. Top canonical pathways associated with significantly differentially expressed genes during liver regeneration in (g) LSECs and (h) HPCs in IPA program. Functional annotations identified using DAVID software with top (i) LSEC and (j) HPC genes selected by evaluating the relative contribution of each gene to differential gene expression during the process of liver regeneration in PLS-DA modeling. (k) Expression of Orm1 and Orm2 in LSECs and HPCs during liver regeneration measured by CAGE analysis.

Article Snippet: For gain-of-function analysis, 24 h after siRNA transfection, FLC4 cells were treated with recombinant human Orm1 (CLPRO889, Cedarlane, Burlington, NC, USA) in DMEM containing 2% FBS for 24 h before cell viability determination.

Techniques: Expressing, Control, Functional Assay, Software, Gene Expression

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Regulatory role of Orm1 on the proliferation of mouse regenerating HPCs and human hepatic cells. (a) Schematic overview of loss-of-function experimental procedures. Orm1 expression at transcript levels in (b) whole-liver tissue and (c) serum protein levels. (d) IHC staining and (e) gene expression of cell proliferation marker Ki-67 in whole-liver tissue at 48 h post-PH. The average percentage of Ki-67 positive cells presented in (d) was quantified from five randomly selected areas in three slides from each mouse (n = 2–4). Scale bar, 50 μm. siCtl, control siRNA; siOrm1, Orm1 siRNA. The quantitative data were presented as the mean plus standard error (n = 4). (f) Data mining of Orm1 expression in human tissues obtained from the freely available FANTOM5 database. Reference RNA, commercially obtained universal human reference total RNA. Others, other human hepatic non-parenchymal cells, primary cells, tissues and cancer cell lines. (g) The gene expression of Orm1 was examined in human liver cancer cell lines FLC4, FLC7, and HepG2, human hepatic stellate cell line LX2, and immortalized human liver endothelial cell line M1 using real-time RT-PCR. Gene expression was normalized to that of GAPDH. Effect of Orm1 knockdown on (h) gene expression of Orm1 and cyclin D1 and (i) cell proliferation of human hepatic cell line FLC4 in the in the absence [ORM1(−)] and presence [ORM1(+)] of 25 ng/mL recombinant human Orm1. The quantitative data were presented as the mean plus the standard deviation of three replicates. * P < 0.05 assessed using two-tailed Student's t -test, Mann-Whitney U test or ANOVA with post-hoc Tukey HSD Calculator for multiple comparison.

Article Snippet: For gain-of-function analysis, 24 h after siRNA transfection, FLC4 cells were treated with recombinant human Orm1 (CLPRO889, Cedarlane, Burlington, NC, USA) in DMEM containing 2% FBS for 24 h before cell viability determination.

Techniques: Expressing, Immunohistochemistry, Gene Expression, Marker, Control, Quantitative RT-PCR, Knockdown, Recombinant, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Molecular targets of Orm1 in mouse regenerating livers. Microarray analysis ( GSE83733 ) was performed in control siRNA (siCtl) and Orm1 siRNA (siOrm1)-injected mouse livers at 48 h post-PH (n = 2). (a) Heatmap visualization of 188 differentially expressed genes with a fold change of > 2 in the livers between groups of mice receiving siOrm1 or siCtl. (b) Top five associated signaling pathways performed using SEA analysis in GeneSpring GX13. (c) The top diseases or functions annotation and (d) top canonical pathway analysis performed in IPA platform. The pathways were ranked according to their-log10 of P values. The ratio indicates the number of enriched genes of interest relative to the total number of genes associated with that pathway in the IPA database. (e) Gene expression levels of MCM2 , MCM4 and MCM6 involved in the enriched “cell cycle control of chromosomal replication” singling pathway in whole-liver tissues before (PH 0 h) and at 48 h post-PH (PH 48 h) were verified using RT-PCR and presented as fold change compared to PH 0 h (n = 4). The data were presented in a Box-and-Whisker plot. * P < 0.05 assessed using the Mann-Whitney U test or two-tailed Student's t -test.

Article Snippet: For gain-of-function analysis, 24 h after siRNA transfection, FLC4 cells were treated with recombinant human Orm1 (CLPRO889, Cedarlane, Burlington, NC, USA) in DMEM containing 2% FBS for 24 h before cell viability determination.

Techniques: Microarray, Control, Injection, Protein-Protein interactions, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Whisker Assay, MANN-WHITNEY, Two Tailed Test

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Schematic diagram of a regulatory role of Orm1 on regenerating HPC proliferation. In this study, beginning with the transcriptome profiling of LSECs and HPCs during mouse liver regeneration, early transcriptional changes in injury response pathways in LSECs was observed, followed by activation of cell cycle control pathways in HPCs, and Orm1, predominantly expressed in HPCs, was found and characterized as a promising factor responsible for that by regulating HPC proliferation during liver regeneration.

Article Snippet: For gain-of-function analysis, 24 h after siRNA transfection, FLC4 cells were treated with recombinant human Orm1 (CLPRO889, Cedarlane, Burlington, NC, USA) in DMEM containing 2% FBS for 24 h before cell viability determination.

Techniques: Activation Assay, Control