Cell Pellets Research Articles

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis.

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

Production of Zinc and Copper as Nanoparticles by Green Synthesis Using Pseudomonas fluorescens.

<b>Background and Objective:</b> Nanoparticles with a little size to an enormous surface (1-100 nm) have expected clinical, mechanical and agricultural applications. This study aimed to produce nano Zinc Oxide (ZnO) and nano Copper Oxide (CuO) particles by green synthesis. <b>Materials and Methods:</b> Two strains of <i>Pseudomonas fluorescens</i> i.e., PSI and PSII, both cell culture supernatants and cell pellets from the two strains were examined separately in CuSO<sub>4</sub> or ZnSO<sub>4</sub> solutions. The supernatants from both strains produced color changes in both solutions referring to the formation of nano CuO or ZnO particles. The solutions were examined for nano-particle characteristics using UV-spectroscopy, particle size and morphology were tested using a scanning electron microscope and transmission electron microscopy. <b>Results:</b> UV-Vis absorption spectrum of solutions at a wavelength range 200-800 nm exhibits a distinct absorption peak in the region of 238-331 and at 303-366 nm for CuO or ZnO NPs, respectively. Absorption bands and the characteristic Surface Plasmon Resonance (SPR) spectra confirm the existence of CuO and ZnO NPs. SEM analysis micrographs indicated that CuO NPs were formed as spherical particles, while the exact shape of ZnO NPs could be identified as oval aggregates. <b>Conclusion:</b> Changes of color occurred in both solutions of two strains referring to the formation of nano CuO or ZnO particles.

Musashi1 inhibit the release of Newcastle disease viruses through preventing apoptosis of DF-1 cells

The efficient proliferation of Newcastle disease virus (NDV) depends on its inhibition of host cell innate immunity. V protein acts as a nonstructural protein which plays a significant role in virus replication, whereas its function remains to be further explored. In this study, Musashi RNA binding protein 1 (MSI1) was selected and its interaction with V protein was further verified by Co-immunoprecipitation (Co-IP) and Immuno-colocalization test. Through the transfection of pCMV-HA-MSI1 in DF-1 cells, the overexpression of MSI1 reduced virus particles in the cell supernatant but not reduced mRNA and virus protein in cells pellet, which suggests that MSI1may act as a new antiviral molecule by inhibiting viral release. Cell early apoptosis was detected by flow cytometry (FCM), the result shows that overexpression of MSI1 inhibit cell apoptosis, implying MSI1 Inhibit virus release may through this way. Taken together, MSI1 and NDV V protein has a detectable interaction, and may block apoptosis to inhibit the release of NDV. However, this is the first report about the interaction between MSI1 and V protein of NDV that can inhibit the NDV replicated.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14days posttransplant. After 75min of kidney warm ischemia, four experimental groups of n=7 underwent 14h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240min of NMP with infusion of vehicle, 10million porcine, or 10million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.

Characteristics of the MACS/WIHS Combined Cohort Study: Opportunities for Research on Aging With HIV in the Longest US Observational Study of HIV.

In 2019, the National Institutes of Health combined the Multicenter AIDS Cohort Study (MACS) and the Women's Interagency HIV Study (WIHS) into the MACS/WIHS Combined Cohort Study (MWCCS). In this paper, participants who made a study visit during October 2018-September 2019 (targeted for MWCCS enrollment) are described by human immunodeficiency virus (HIV) serostatus and compared with people living with HIV (PLWH) in the United States. Participants include 2,115 women and 1,901 men with a median age of 56 years (interquartile range, 48-63); 62% are PLWH. Study sites encompass the South (18%), the Mid-Atlantic/Northeast (45%), the West Coast (22%), and the Midwest (15%). Participant race/ethnicity approximates that of PLWH throughout the United States. Longitudinal data and specimens collected for 35 years (men) and 25 years (women) were combined. Differences in data collection and coding were reviewed, and key risk factor and comorbidity data were harmonized. For example, recent use of alcohol (62%) and tobacco (28%) are common, as are dyslipidemia (64%), hypertension (56%), obesity (42%), mildly or severely impaired daily activities (31%), depressive symptoms (28%), and diabetes (22%). The MWCCS repository includes serum, plasma, peripheral blood mononuclear cells, cell pellets, urine, cervicovaginal lavage samples, oral samples, B-cell lines, stool, and semen specimens. Demographic differences between the MACS and WIHS can confound analyses by sex. The merged MWCCS is both an ongoing observational cohort study and a valuable resource for harmonized longitudinal data and specimens for HIV-related research.

Diagnostic performance of an ultrasensitive HRP2-based malaria rapid diagnostic test kit used in surveys of afebrile people living in Southern Ghana

BackgroundThe Alere™ Malaria Ag P.f Ultra-sensitive RDT (UsmRDT) kit is an HRP2-based malaria rapid diagnostic test (RDT) with enhanced sensitivity relative to the SD Bioline Malaria Ag P.f RDT (mRDT) kit. However, the diagnostic performance of the UsmRDT kit has not been evaluated in Ghana.MethodsA total of 740 afebrile participants aged between 3 and 88 years old were recruited from the Central and Greater Accra Regions of Ghana during the off-peak malaria season. Axillary body temperature was measured, and a volume of 1 ml venous blood was drawn from each participant. Prior to separating the blood into plasma and packed cell pellets via centrifugation, the blood was spotted onto one UsmRDT and one mRDT kit and also used to prepare thick and thin blood smears as well as filter paper blood spots. Plasmodium falciparum specific polymerase chain reaction (PCR) was performed on gDNA extracted from 100 µl of the whole blood.ResultsThe overall positivity rate for microscopy, PCR, UsmRDT and mRDT kit were 20.4%, 40.8%, 31.3% and 30.8%, respectively. Overall, the UsmRDT identified 9.3% (28/302) more PCR positive samples than the mRDT kits. All samples that were negative by the UsmRDT kit were also negative by the mRDT kit. Overall, the sensitivity and specificity of the UsmRDT was 73% (221/302) and 89% (388/436), respectively, while that for the mRDT kit was 58% and 90%, respectively.ConclusionAlthough the UsmRDT kit was not as sensitive as PCR at detecting asymptomatic P. falciparum carriage, it correctly identified P. falciparum in 9.3% of the study participants that were not captured by the mRDT kit. In malaria endemic settings, the UsmRDT would provide an added advantage by identifying more asymptomatic P. falciparum carriers than the mRDT kit for targeted treatment interventions.

Genomics assisted functional characterization of Paenibacillus polymyxa HK4 as a biocontrol and plant growth promoting bacterium

The diseases caused by phytopathogens account for huge economic losses in the agricultural sector. Paenibacillus polymyxa is one of the agriculturally important biocontrol agents and plant growth promoting bacterium. This study describes the antifungal potential of P. polymyxa HK4 against an array of fungal phytopathogens and its ability to stimulate seed germination of cumin and groundnut under in vitro conditions. The cumin and groundnut seeds bacterized with HK4 exhibited enhanced germination efficiency in comparison to controls. The use of HK4 as a soil inoculant significantly promoted the shoot length and fresh weight of groundnut plants in pot studies. The draft genome analysis of HK4 revealed the genetic attributes for motility, root colonization, antagonism, phosphate solubilization, siderophore production and production of volatile organic compounds. The bacterium HK4 harnessed several hydrolytic enzymes that may assist its competence in the rhizosphere. The PCR amplification and sequence analysis of the conserved region of the fusA gene amplicon revealed the ability of HK4 to produce fusaricidin. Furthermore, the LC-ESI-MS/MS of crude cell pellet extract of HK4 confirmed the presence of fusaricidin as a major antifungal metabolite. This study demonstrated the potential of HK4 as a biocontrol agent and a plant growth promoter.

Embedding Cultured Cells in Matrigel for Staining.

Adherent cells normally are prepared for cell staining by growing on a suitable support. Suspension cells can be fixed directly or can be attached to a solid support by centrifugation, chemical cross-linking, or simple dehydration. An alternative approach for cells grown in culture is to prepare a fixed cell pellet that is embedded in a scaffold, such as Matrigel, and then processed, embedded in paraffin, and stained in a manner identical to formalin-fixed tissue specimens.

Compressive mechanical stress enhances susceptibility to interleukin-1 by increasing interleukin-1 receptor expression in 3D-cultured ATDC5 cells

BackgroundMechanical overload applied on the articular cartilage may play an important role in the pathogenesis of osteoarthritis. However, the mechanism of chondrocyte mechanotransduction is not fully understood. The purpose of this study was to assess the effects of compressive mechanical stress on interleukin-1 receptor (IL-1R) and matrix-degrading enzyme expression by three-dimensional (3D) cultured ATDC5 cells. In addition, the implications of transient receptor potential vanilloid 4 (TRPV4) channel regulation in promoting effects of compressive mechanical loading were elucidated.MethodsATDC5 cells were cultured in alginate beads with the growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. The cultured cell pellet was seeded in collagen scaffolds to produce 3D-cultured constructs. Cyclic compressive loading was applied on the 3D-cultured constructs at 0.5 Hz for 3 h. The mRNA expressions of a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS4) and IL-1R were determined with or without compressive loading, and effects of TRPV4 agonist/antagonist on mRNA expressions were examined. Immunoreactivities of reactive oxygen species (ROS), TRPV4 and IL-1R were assessed in 3D-cultured ATDC5 cells.ResultsIn 3D-cultured ATDC5 cells, ROS was induced by cyclic compressive loading stress. The mRNA expression levels of ADAMTS4 and IL-1R were increased by cyclic compressive loading, which was mostly prevented by pyrollidine dithiocarbamate. Small amounts of IL-1β upregulated ADAMTS4 and IL-1R mRNA expressions only when combined with compressive loading. TRPV4 agonist suppressed ADAMTS4 and IL-1R mRNA levels induced by the compressive loading, whereas TRPV4 antagonist enhanced these levels. Immunoreactivities to TRPV4 and IL-1R significantly increased in constructs with cyclic compressive loading.ConclusionCyclic compressive loading induced mRNA expressions of ADAMTS4 and IL-1R through reactive oxygen species. TRPV4 regulated these mRNA expressions, but excessive compressive loading may impair TRPV4 regulation. These findings suggested that TRPV4 regulates the expression level of IL-1R and subsequent IL-1 signaling induced by cyclic compressive loading and participates in cartilage homeostasis.

Exopolysaccharides Production by Cultivating a Bacterial Isolate from the Hypersaline Environment of Salar de Uyuni (Bolivia) in Pretreatment Liquids of Steam-Exploded Quinoa Stalks and Enzymatic Hydrolysates of Curupaú Sawdust

The halotolerant bacterial strain BU-4, isolated from a hypersaline environment, was identified as an exopolysaccharide (EPS) producer. Pretreatment liquids of steam-exploded quinoa stalks and enzymatic hydrolysates of Curupaú sawdust were evaluated as carbon sources for EPS production with the BU-4 strain, and the produced EPS was characterized using FTIR, TGA, and SEM. Cultivation was performed at 30 °C for 48 h, and the cells were separated from the culture broth by centrifugation. EPS was isolated from the cell pellets by ethanol precipitation, and purified by trichloroacetic acid treatment, followed by centrifugation, dialysis, and freeze-drying. EPS production from quinoa stalks- and Curupaú sawdust-based substrates was 2.73 and 0.89 g L−1, respectively, while 2.34 g L−1 was produced when cultivation was performed on glucose. FTIR analysis of the EPS revealed signals typical for polysaccharides, as well as ester carbonyl groups and sulfate groups. High thermal stability, water retention capacity and gel-forming ability were inferred from SEM and TGA. The capability of the halotolerant isolate for producing EPS from pretreatment liquids and hydrolysates was demonstrated, and characterization of the EPS revealed their broad application potential. The study shows a way for producing value-added products from waste materials using a bacterium from a unique Bolivian ecosystem.

Application of Bio-Friendly Formulations of Chitinase-Producing Streptomyces cellulosae Actino 48 for Controlling Peanut Soil-Borne Diseases Caused by Sclerotium rolfsii.

Of ten actinobacterial isolates, Streptomyces cellulosae Actino 48 exhibited the strongest suppression of Sclerotium rolfsii mycelium growth and the highest chitinase enzyme production (49.2 U L−1 min−1). The interaction between Actino 48 and S. rolfsii was studied by scanning electron microscope (SEM), which revealed many abnormalities, malformations, and injuries of the hypha, with large loss of S. rolfsii mycelia density and mass. Three talc-based formulations with culture broth, cell-free supernatant, and cell pellet suspension of chitinase-producing Actino 48 were characterized using SEM, Fourier transform infrared spectroscopy (FTIR), and a particle size analyzer. All formulations were evaluated as biocontrol agents for reducing damping-off, root rot, and pods rot diseases of peanut caused by S. rolfsii under greenhouse and open-field conditions. The talc-based culture broth formulation was the most effective soil treatment, which decreased the percentage of peanut diseases under greenhouse and open-field conditions during two successive seasons. The culture broth formulation showed the highest increase in the dry weight of peanut shoots, root systems, and yielded pods. The transcriptional levels of three defense-related genes (PR-1, PR-3, and POD) were elevated in the culture broth formulation treatment compared with other formulations. Subsequently, the bio-friendly talc-based culture broth formulation of chitinase-producing Actino 48 could potentially be used as a biocontrol agent for controlling peanut soil-borne diseases caused by S. rolfsii.

Bacillus subtilis L1-21 as a biocontrol agent for postharvest gray mold of tomato caused by Botrytis cinerea

Gray mold (Botrytis cinerea) is a serious disease causing considerable losses to harvested tomato during storage and transportation. Extensive and unjudicial use of fungicides has certain deleterious effects such as evolved resistance in pathogen and environmental pollution. The ice cold chain transportation is the most commonly used method, but the results are not satisfactory. Currently, the trend is shifting towards prevention and control measures which ensures environmental protection, therefore, the biological control of postharvest diseases has become a hot spot. In this study, Bacillus subtilis L1-21 was used to control gray mold on tomato fruits, by adopting plate confrontation and fruit acupuncture method. To evaluate the preservative effect of biocontrol agent L1-21 on tomato, the gfp-tagged L1-21 was used to study colonization potential in harvested tomatoes assay. Colonization of L1-21-gfp in postharvest tomatoes was stable at 103 cfu/g by soaking and fluorescence method. Whereas inhibition rate of B. cinerea was 39.63% ± 4.73% in plate confrontation. Culture suspension of L1-21 at a concentration of 108 cfu/ml displayed the best control effect: 86.57% ± 2.55% against tomato gray mold. In addition, different components exhibited different results such as cell pellet + saline (72.94% ± 8.49%), supernatant (41.18% ± 16.22%), sterilized culture (28.56% ± 6.54%). Further, suspension of B. cinerea and Bacillus L1-21 injected in tested tomatoes led to maximum control effect (81.40% ± 2.74%) after 4 h. Injection of L1-21 culture into tomato followed by pathogen B. cinerea spore solution resulted in 100%±0% control effect after 48 h. There was no significant difference in fruit quality between treated and untreated tomatoes. Taken together, B. subtilis L1-21 may lay theoretical foundation to be used in prevention of gray mold and other postharvest diseases during storage time.

Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

BackgroundMicrobial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host.ResultsAnalysis of intra- and extracellular changes identified 2380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain.ConclusionsThis is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

New insights into molecular aspects of sperm–oviductal binding in Egyptian buffaloes using anin vitromodel: Effects of oviductal segments and media

We aimed (I) to simulate an in vivo milieu, through establishing an in vitro paradigm to study sperm-oviductal interactions using different segments of oviduct, as well as different incubation media, and (II) to investigate spatial changes of oviductal gene expression. Two experiments were designed; one was to investigate the yield of oviduct aggregates from different oviduct segments; in the second experiment, we observed effects of different incubation media on sperm-oviductal binding. Oviduct cell pellets before (control) and after sperm binding were collected for RNA isolation and gene expression. Isthmus resulted in a higher aggregate yield and possessed the highest affinity towards spermatozoa. The different segments of oviduct showed clear changes in gene expression after sperm binding. TALP medium promoted formation of a higher number of oviduct aggregates towards spermatozoa. Different media resulted in profound alterations in isthmus gene expression. Collectively, isthmus segment in TALP media showed the highest binding affinity to spermatozoa. At the molecular level, our in vitro model was successful for simulation in vivo milieu. Thus, our findings could be used as a simple tool to gain more insights into the molecular regulation of sperm movement, selection and affinity for oviductal binding in buffaloes.

Centrifugation does not remove bacteria from the fat fraction of human milk

Analysis of the human milk microbiome is complicated by the presence of a variable quantity of fat. The fat fraction of human milk is typically discarded prior to analysis. It is assumed that all cells are pelleted out of human milk by high speed centrifugation; however, studies of bovine milk have reported that bacteria may remain trapped within the fat fraction. Here, the bacterial DNA profiles of the fat fraction and cell pellet of human milk (n = 10) were analysed. Human and bacterial DNA was consistently recovered from the fat fraction of human milk (average of 12.4% and 32.7%, respectively). Two low-abundance Staphylococcus species (< 0.5% relative abundance) was significantly more abundant in the cell pellet compared to the fat fraction (P < 0.04), and three low-abundance species (< 5% relative abundance) were recovered from one fraction only. However, inclusion of fat reduced the efficiency of DNA extraction by 39%. Culture-based methods were used to quantify the distribution of an exogenously added strain of Staphylococcus aureus in human milk fractions. S. aureus was consistently recovered from the fat fraction (average 28.9%). Bacterial DNA profiles generated from skim milk or cell pellets are not representative of the entire human milk microbiome. These data have critical implications for the design of future work in this field.

Growth kinetics of a Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture.

Growth kinetics of a Vero cells adapted Bangladeshi strain of peste des petits ruminants virus was studied in Vero cells to determine maximum virus yield. One-step growth curve was formulated after determining virus in both supernatant (CFV) and cell lysate (CAV) at different time categories by microtitre plate titration in Vero cells and the viral presence was confirmed by real-time RT-PCR. The virus was first detected in both the supernatants and cell pellets at 12 hpi. The virus titre reached its plateau at 72 hpi. Maximum virus titre of CAV was 6.2 log10 TCID50/ml and that of CFV was 5.2 log10 TCID50/ml at 72 hpi. After that, the titer gradually declined, but maintained at 4.5 log10 TCID50/ml in case of CAV and 4.2 log10 TCID50/ml in case of CFV at 96 hpi. It was concluded that the optimum time point for harvesting Vero cell culture is 72 hpi.

Quantification of Duloxetine in the Bacterial Culture and Medium to Study Drug-gut Microbiome Interactions.

Expanding our understanding of drug-gut bacteria interactions requires high-throughput drug measurements in complex bacterial cultures. Quantification of drugs in the cultures, media, and cell pellets is prone to strong matrix effects. We have developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for quantifying duloxetine from high-throughput gut-drug interaction experiments. The method is partially validated for its reproducibility, sensitivity, and accuracy, which makes it suitable for largescale drug screens. We extensively used this method to study biotransformation and bioaccumulation of duloxetine and other drugs in several species of gut bacteria.

Tanshinone IIA, a melanogenic ingredient basis of Salvia miltiorrhiza Bunge

Background: The roots of Salvia miltiorrhiza Bunge (Lamiaceae) have been often used to treat vitiligo in clinical for many years. However, the main ingredient basis of efficacy has not been known. Objectives: We investigated whether the two main constituents of S. miltiorrhiza, major hydrophobic compound tanshinone IIA (Tan IIA) and the major hydrophilic compound salvianolic acid B (Sal B), had the same melanogenic activity. Methods: To testify the potential roles of Tan IIA and Sal B in pigmentation, tyrosinase (Tyr) activity, melanin synthesis ability, and the molecular mechanisms stimulating melanin production were determined in B16F10 melanoma cells. Results: Tan IIA promoted melanogenesis and enhanced Tyr activity at its maximum concentration (10 μM), whereas that of Sal B had no effect. Furthermore, the color of cell pellets and morphological observation of B16F10 cells were visibly darkened. Regarding molecular mechanisms, Western blot results showed that Tan IIA (1, 3, and 10 μM) dose dependently increased the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase MAPK, respectively. However, it decreased phosphorylation in extracellular signal-regulated protein kinase 1/2 MAPK signaling. Meanwhile, Tan IIA promoted the expression of microphthalmia-associated transcription factor and Tyr. Conclusion: Tan IIA might be a melanogenic ingredient basis of S. miltiorrhiza to increase the Tyr activity by activating the upstream MAPK signaling pathways, thereby contributing to pigmentary processing.