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Results for “protocol optimization”

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PubMed2004

Efficient transfection of embryonic and adult stem cells.

The ability of embryonic stem cells and adult stem cells to differentiate into specific cell types holds immense potential for therapeutic use in cell and gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. In the study reported here, we demonstrate the use of nucleofection as a method to introduce plasmid DNA into embryonic and adult stem cells with significantly greater efficiency than electroporation or lipid-based transfection methods have. Using enhanced green fluorescent protein (eGFP) as a reporter gene, mouse embryonic stem cells were transfected both transiently and stably at a rate nearly 10-fold higher than conventional methods. The transfected cells retained their stem cell properties, including continued expression of the stem cell markers SSEA1, Oct4, and Rex1; formation of embryoid bodies; differentiation into cardiomyocytes in the presence of appropriate inducers; and, when injected into developing blastocysts, contribution to chimeras. Higher levels of transfection were also obtained with human embryonic carcinoma and human embryonic stem cells. Particularly hard-to-transfect adult stem cells, including bone marrow and multipotent adult progenitor cells, were also transfected efficiently by the method of nucleofection. Based on our results, we conclude that nucleofection is superior to currently available methods for introducing plasmid DNA into a variety of embryonic and adult stem cells. The high levels of transfection achieved by nucleofection will enable its use as a rapid screening tool to evaluate the effect of ectopically expressed transcription factors on tissue-specific differentiation of stem cells.

Animals↗

PubMed2002

High-quality RNA from cells isolated by laser capture microdissection.

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.

Animals↗

PubMed2002

Optimized sample preparation for tandem capillary electrophoresis single-stranded conformational polymorphism/ heteroduplex analysis.

Here we describe DNA sample preparation methods that allow the rapid, simultaneous generation of both single-stranded conformational polymorphism (SSCP) and heteroduplex DNA elements from a single sample in a single tube, which are suitable for direct injection into a capillary electrophoresis (CE) instrument with excellent sensitivity of genetic mutation detection. The p53 gene was used as a model DNA region for this study, which was performed on a high-throughput MegaBACE 96-capillary array electrophoresis instrument. We found that, contrary to the practice common in slab-gel SSCP analysis, denaturants such as formamide are incompatible with this novel technique because they result in homo- and heteroduplex peak broadening in CE (possibly as a result of incomplete dsDNA re-hybridization) that reduces the peak resolution and hence the sensitivity of mutation detection. We also have found that PCR buffers, which are typically used to suspend samples for slab-gel heteroduplex analysis (HA), but which are less suitable for CE because of the presence of extra salt that reduces the efficiency of electrokinetic injection, may be substituted with a 10 mM Tris-HCI buffer (pH 8.5). The use of this Tris-HCl buffer for sample preparation provides both a high sensitivity of mutation detection by tandem SSCP/HA and high efficiency ofelectrokinetic injection by CE. In a related study (published elsewhere), we have applied this optimized protocol to the screening of a set of 32 mutant DNA samples from p53 exons 7 and 8 and recorded 100% sensitivity of mutation detection for tandem CE-SSCP/HA, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA).

DNA↗

PubMed2003

The impact of resistance training on distance running performance.

Traditionally, distance running performance was thought to be determined by several characteristics, including maximum oxygen consumption (VO(2max)), lactate threshold (LT), and running economy. Improvements in these areas are primarily achieved through endurance training. Recently, however, it has been shown that anaerobic factors may also play an important role in distance running performance. As a result, some researchers have theorised that resistance training may benefit distance runners. Because resistance training is unlikely to elicit an aerobic stimulus of greater than 50% of VO(2max), it is unlikely that resistance training would improve VO(2max) in trained distance runners. However, it appears that VO(2max) is not compromised when resistance training is added to an endurance programme. Similarly, LT is likely not improved as a result of resistance training in trained endurance runners; however, improvements in LT have been observed in untrained individuals as a result of resistance training. Trained distance runners have shown improvements of up to 8% in running economy following a period of resistance training. Even a small improvement in running economy could have a large impact on distance running performance, particularly in longer events, such as marathons or ultra-marathons. The improvement in running economy has been theorised to be a result of improvements in neuromuscular characteristics, including motor unit recruitment and reduced ground contact time. Although largely theoretical at this point, if resistance training is to improve distance running performance, it will likely have the largest impact on anaerobic capacity and/or neuromuscular characteristics. The primary purpose of this review is to consider the impact of resistance training on the factors that are known to impact distance running performance. A second purpose is to consider different modes of resistance exercise to determine if an optimal protocol exists.

Anaerobiosis↗

PubMed2006

Abdominal MRI at 3.0 T: the basics revisited.

OBJECTIVE: The purpose of our article is to describe the underlying physics concepts of abdominal MRI at 3.0 T and their impact on signal-to-noise ratio, susceptibility artifacts, chemical shift artifacts, and dielectric effects. CONCLUSION: Abdominal MR sequence protocols optimized for 1.5-T scanners should not be transferred to 3.0 T without substantial modification. In addition, specific patient groups--for example, large patients with ascites--are not well suited to undergo an abdominal MRI study at 3.0 T.

Abdomen↗

PubMed2004

Temporary preservation of beta-cell function by diazoxide treatment in childhood type 1 diabetes.

OBJECTIVE: We examined the effect of diazoxide, an ATP-sensitive K(+) channel opener and inhibitor of insulin secretion, on beta-cell function and remission in children at clinical onset of type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 56 subjects (21 girls and 35 boys, age 7-17 years) were randomized to 3 months of active treatment (diazoxide 5-7.5 mg/kg in divided doses) or placebo in addition to multiple daily insulin injections and were followed for 2 years. RESULTS: Diazoxide decreased circulating C-peptide concentrations by approximately 50%. After cessation of the treatment, basal and meal-stimulated C-peptide concentrations increased to a maximum at 6 months, followed by a decline. Meal-stimulated C-peptide concentration was significantly higher at 12 months (0.43 +/- 0.22 vs. 0.31 +/- 0.26 nmol/l, P = 0.018) and tended to fall less from clinical onset to 24 months in the diazoxide- vs. placebo-treated patients (-0.05 +/- 0.24 vs. -0.18 +/- 0.26 nmol/l, P = 0.064). At 24 months, the meal-stimulated C-peptide concentrations were 0.24 +/- 0.20 and 0.20 +/- 0.17 nmol/l, respectively. Side effects of diazoxide were prevalent. CONCLUSIONS: This study demonstrates that partial inhibition of insulin secretion for 3 months at onset of childhood type 1 diabetes suspends the period of remission and temporarily preserves residual insulin production. Further evaluation of the full potential of beta-cell rest will require compounds with less side effects as well as protocols optimized for sustained secretory arrest.

Adolescent↗

PubMed2001

Fluorophotometric determination of aqueous humor flow rate in clinically normal dogs.

OBJECTIVE: To determine aqueous humor flow rate in clinically normal dogs, using fluorophotometry. ANIMALS: 20 clinically normal Beagles. PROCEDURE: A study was performed on 5 dogs to establish an optimal protocol for fluorophotometric determination of aqueous humor flow rate. This protocol then was used to measure aqueous humor flow rate in 15 dogs. Corneas were loaded with fluorescein by topical application, and corneal and aqueous humor fluorescein concentrations were measured 5, 6.5, and 8 hours after application. Concentration-versus-time plots were generated, and slopes and ratios of the fluorescein concentration in the cornea and aqueous humor from these graphs were used to calculate flow rates. Calculations were performed by use of automated software provided with the fluorophotometer and by manual computation, and the 2 calculation methods were compared. RESULTS: The protocol established for the 5 dogs resulted in semilogarithmic and parallel decay of corneal and aqueous humor concentrations. Manually calculated mean +/- SD aqueous humor flow rates for left, right, and both eyes were 5.58 +/- 2.42, 4.86 +/- 2.49, and 5.22 +/- 1.87 microl/min, respectively, whereas corresponding flow rates calculated by use of the automated software were 4.54 +/- 3.08, 4.54 +/- 3.10, and 4.54 +/- 2.57 microl/min, respectively. Values for the left eye were significantly different between the 2 computation methods. CONCLUSIONS AND CLINICAL RELEVANCE: Aqueous humor flow rates can be determined in dogs, using fluorophotometry. This technique can be used to assess pathologic states and medical and surgical treatments that alter aqueous humor dynamics.

Animals↗

PubMed1999

Evaluation of slice shear force as an objective method of assessing beef longissimus tenderness.

Experiments were conducted to develop an optimal protocol for measurement of slice shear force (SSF) and to evaluate SSF as an objective method of assessing beef longissimus tenderness. Whereas six cylindrical, 1.27-cm-diameter cores are typically removed from each steak for Warner-Bratzler shear force (WBSF) determination, a single 1-cm-thick, 5-cm-long slice is removed from the lateral end of each longissimus steak for SSF. For either technique, samples are removed parallel to the muscle fiber orientation and sheared across the fibers. Whereas WBSF uses a V-shaped blade, SSF uses a flat blade with the same thickness (1.016 mm) and degree of bevel (half-round) on the shearing edge. In Exp. 1, longissimus steaks were acquired from 60 beef carcasses to determine the effects of belt grill cooking rate (very rapid vs. rapid) and conditions of SSF measurement (hot vs cold) on the relationship of SSF with trained sensory panel (TSP) tenderness rating. Slice shear force was more strongly correlated with TSP tenderness rating when SSF measurement was conducted immediately after cooking (r = -.74 to -.76) than when steaks were chilled (24 h, 4 degrees C) before SSF measurement (r = -.57 to -.72). When SSF measurement was conducted immediately after cooking, the relationship of SSF with TSP tenderness rating did not differ among the belt grill cooking protocols used to cook the SSF steak. In Exp. 2, longissimus steaks were acquired from 479 beef carcasses to compare the ability of SSF and WBSF of 1.27-cm-diameter cores to predict TSP tenderness ratings. Slice shear force was more strongly correlated with sensory panel tenderness rating than was WBSF (r = -.82 vs -.77). In Exp. 3, longissimus steaks were acquired from 110 beef carcasses to evaluate the repeatability (.91) of SSF over a broad range of tenderness. Slice shear force is a more rapid, more accurate, and technically less difficult technique than WBSF. Use of the SSF technique could facilitate the collection of more accurate data and should allow the detection of treatment differences with reduced numbers of observations and reduced time requirements, thereby reducing research costs.

Animals↗

PubMed1987

T cell depletion of bone marrow for clinical marrow allografting: optimalization of conditions for depletion by anti-CD2 and anti-CD8 monoclonal antibodies with rabbit complement, and for detection of residual T cell content.

Normal bone marrow obtained at harvest for bone marrow transplantation was passed over a Ficoll density gradient to obtain the mononuclear cell fraction. These cells were incubated with anti-T cell monoclonal antibodies (MAb) anti-HuLym-1 and anti-HuLym-8 plus rabbit complement, washed, and the degree of T cell depletion was assessed by culture in phytohemagglutinin, by flow cytometry and by limiting dilution analysis. The optimal protocol for T cell depletion was found to be a 2-stage incubation with 2 cycles of complement treatment, using a bone marrow cell concentration as low as possible. The precise conditions of complement dilution and incubation temperature depended on the batch of complement used, and had to be assessed for each batch. Of the 3 methods used for assessing T cell contamination, culture in PHA was found to be inappropriate due to large variation in background proliferation of treated and untreated bone marrow cells. Flow cytometry was accurate for residual T cell content of greater than 1% (corresponding to a 90-95% depletion of T cells) but could not detect lower levels of T cell contamination. The limit dilution assay was found to be by far the most sensitive, being capable of detecting a T cell contamination of 1 in 10,000 cells. When combined with flow cytometry on untreated marrow for calculation of the cloning efficiency, it gave very accurate determinations of residual T cell content of marrow.

Animals↗

PubMed1999

Ligaments and tendons of the ankle. Evaluation with low-field (0.2 T) MR imaging.

PURPOSE: To investigate the value of MR imaging using a low-field imaging unit (0.2 T) for the evaluation of ligaments and tendons of the ankle. MATERIAL AND METHODS: Twelve ankle specimens were studied using low-field MR imaging (0.2 T). The Achilles tendon, the tibialis posterior tendon, and the lateral collateral ligaments were evaluated for the presence of degenerative changes and partial and complete tears. Visibility and overall image quality were analyzed by qualitative evaluation. RESULTS: Low-field MR images of the 24 tendons were interpreted as normal in 11 cases, showing degenerative changes in 9 cases and a partial tear in 1 case. Visualization was regarded as not sufficient for a diagnosis in 3 cases. Of the 36 ligaments, 14 were regarded as normal while degenerative changes were seen in 5 cases and a tear in 2 cases. In 13 cases, no diagnosis could be established, and in 2 cases only a probable diagnosis was established. The best overall quality was obtained with the use of T1 spin-echo (680/20/4) and T2 multi-echo (3000/40/1) images. CONCLUSION: Optimized protocols for examination of the ankle using low-field MR imaging may allow evaluation of the Achilles and tibialis posterior tendons, but these protocols may not be as useful for diagnosing ligamentous changes.

Achilles Tendon↗

PubMed2000

In situ detection of a PCR-synthesized human pancentromeric DNA hybridization probe by color pigment immunostaining: application for dicentric assay automation.

We report a low cost and efficient method for synthesizing a human pancentromeric DNA probe by the polymerase chain reaction (PRC) and an optimized protocol for in situ detection using color pigment immunostaining. The DNA template used in the PCR was a 2.4 kb insert containing human alphoid repeated sequences of pancentromeric DNA subcloned into pUC9 (Miller et al. 1988) and the primers hybridized to internal sequences of the 172 bp consensus tandem repeat associated with human centromeres. PCR was performed in the presence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spreads. Detection of pancentromeric probe was achieved by immunoenzymatic color pigment painting to yield a permanent image detected at high resolution by bright field microscopy. The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enhanced identification and eventually to automation of various chromosome aberration assays.

Automation↗

PubMed1994

Permeabilization, staining and culture of living Drosophila embryos.

The organic solvent octane has been used routinely to permeabilize the hydrophobic vitelline membrane surrounding the Drosophila embryo, thereby allowing the movement of small molecules into the egg. We present evidence that hexane is a more effective permeabilizing agent than octane and compare the effects of these solvents on uniformity of permeabilization and embryonic viability. The ability of each solvent to make the embryo accessible to a range of biological stains was compared. The effect of octane versus hexane permeabilization on subsequent embryonic viability was measured at seven different stages during early embryogenesis. We found that although hexane is a superior solvent for permeabilizing the vitelline membrane, it decreases the viability of embryos exposed between 0 and 3 hr of age. Older embryos treated with either hexane or octane are usually viable. We also showed that molecules with a molecular mass of 984 Daltons or more did not diffuse into the embryo following treatment with either hexane or octane. Results presented here challenge a phase-partition model that has been proposed previously to explain the molecular basis of permeabilization of the Drosophila egg. An alternative model is described as well as an optimized protocol for permeabilizing and staining Drosophila embryos at any stage during early embryogenesis while maintaining viability for subsequent culture.

Animals↗

PubMedDate not supplied

Artificial intelligence techniques for cancer treatment planning.

An artificial intelligence system, NEWCHEM, for the development of new oncology therapies is described. This system takes into account the most recent advances in molecular and cellular biology and in cell-drug interaction, and aims to guide experimentation in the design of new optimal protocols. Further work is being carried out, aimed to embody in the system all the basic knowledge of biology, physiopathology and pharmacology, to reason qualitatively from first principles so as to be able to suggest cancer therapies.

Animals↗

PubMed2004

Minimally invasive delivery of stem cells for spinal cord injury: advantages of the lumbar puncture technique.

OBJECT: Stem cell therapy has been shown to have considerable therapeutic potential for spinal cord injuries (SCIs); however, most experiments in animals have been performed by injecting cells directly into the injured parenchyma. This invasive technique compromises the injured spinal cord, although it delivers cells into the hostile environment of the acutely injured cord. In this study, the authors tested the possibility of delivering stem cells to injured spinal cord by using three different minimally invasive techniques. METHODS: Bone marrow stromal cells (BMSCs) are clinically attractive because they have shown therapeutic potential in SCI and can be obtained in patients at the bedside, raising the possibility of autologous transplantation. In this study transgenically labeled cells were used for transplantation, facilitating posttransplantation tracking. Inbred Fisher-344 rats received partial cervical hemisection injury, and 2 x 10(6) BMSCs were intravenously, intraventricularly, or intrathecally transplanted 24 hours later via lumbar puncture (LP). The animals were killed 3, 10, or 14 days posttransplantation, and tissue samples were submitted to histochemical and immunofluorescence analyses. For additional comparison and validation, lineage restricted neural precursor (LRNP) cells obtained from E13.5 rat embryos were transplanted via LP, and these findings were also analyzed. CONCLUSIONS: Both BMSCs and LRNP cells home toward injured spinal cord tissues. The use of LP and intraventricular routes allows more efficient delivery of cells to the injured cord compared with the intravenous route. Stem cells delivered via LP for treatment of SCI may potentially be applicable in humans after optimal protocols and safety profiles are established in further studies.

Animals↗

PubMedDate not supplied

Optimization of wet radiofrequency ablation using a perfused-cooled electrode: a comparative study in ex vivo bovine livers.

OBJECTIVE: To determine the optimized protocol for wet monopolar radiofrequency ablation (RFA) using a perfused-cooled electrode to induce coagulation necrosis in the ex vivo bovine liver. MATERIALS AND METHODS: Radiofrequency was applied to excised bovine livers in a monopolar mode using a 200W generator with an internally cooled electrode (groups A and B) or a perfused-cooled electrode (groups C, D, E, and F) at maximum power (150-200 W) for 10 minutes. A total of 60 ablation zones were created with six different regimens: group A-dry RFA using intra-electrode cooling; group B-dry RFA using intra-electrode cooling and a pulsing algorithm; group C-wet RFA using only interstitial hypertonic saline (HS) infusion; group D-wet RFA using interstitial HS infusion and a pulsing algorithm; group E-wet RFA using interstitial HS infusion and intra-electrode cooling; and group F-wet RFA using interstitial HS infusion, intra-electrode cooling and a pulsing algorithm. In groups C, D, E, and F, RFA was performed with the infusion of 6% HS through the perfused cooled electrode at a rate of 2 mL/minute. During RFA, we measured the tissue temperature at a distance of 15 mm from the electrode. The dimensions of the ablation zones and the changes in impedance, currents, and liver temperature during RFA were compared between these six groups. RESULTS: During RFA, the mean tissue impedances in groups A (243+/-88 ohm) and C (252.5+/-108 ohm) were significantly higher than those in groups B (85+/-18.7 ohm), D (108.2+/-85 ohm), E (70.0+/-16.3 ohm), and F (66.5+/-7 ohm) (p < 0.05). The mean currents in groups E and F were significantly higher than those in groups B and D, which were significantly higher than those in groups A and C (p < 0.05): 520+/-425 mA in group A, 1163+/-34 mA in group B, 652.5+/-418 mA in group C, 842.5+/-773 mA in group D, 1665+/-295 mA in group E, and 1830+/-109 mA in group F. The mean volumes of the ablation regions in groups E and F were significantly larger than those in the other groups (p < 0.05): 17.7+/-5.6 cm(3) in group A, 34.5+/-3.0 cm(3) in group B, 20.2+/-15.6 cm(3) in group C, 36.1+/-19.5 cm(3) in group D, 68.1+/-12.4 cm(3) in group E, and 79.5+/-31 cm(3) in group F. The final tissue temperatures at a distance of 15 mm from the electrode were higher in groups E and F than those in groups A, C, and D (p < 0.05): 50+/-7.5 degrees C in group A, 66+/-13.6 degrees C in group B, 60+/-13.4 degrees C in group C, 61+/-12.7 degrees C in group D, 78+/-14.2 degrees C in group E, and 79+/-12.0 degrees C in group F. CONCLUSION: Wet monopolar RFA, using intra-electrode cooling and interstitial saline infusion, showed better performance in creating a large ablation zone than either dry RFA or wet RFA without intra-electrode cooling.

Animals↗

PubMed1989

Efficacy of fertilization in artificially inseminated turkey hens.

Research was conducted to develop an artificial insemination protocol optimizing the use of spermatozoa by turkey breeder hens. Large White turkey hens were inseminated on Days 14 and 17 postphotostimulation with 200 million spermatozoa from one male phenotype to fill the oviductal storage sites. Artificial inseminations were then performed weekly for 20 wk with different spermatozoa numbers of another male phenotype. Fertility and phenotype of each poult were determined at hatch to ascertain which insemination, initial or subsequent, was responsible for fertility. Inseminating weekly with 200 million viable spermatozoa cells resulted in better fertility but did not optimize the hen's utilization of spermatozoa from the initial inseminations. When fewer spermatozoa were inseminated weekly (50 million cells), more progeny were fertilized by spermatozoa already residing in the oviduct than would be expected. When the number of spermatozoa inseminated weekly was increased at intervals during a laying cycle, spermatozoa from the initial inseminations were utilized more efficiently, but fertility was depressed at times during the laying cycle. Gradually increasing weekly inseminated numbers of spermatozoa from 50 to 200 million viable cells/hen as the hens age results in nearly equivalent fertility to that resulting from insemination by 200 million cells each week. This represents a savings of 1.4 billion spermatozoa/hen over a 20-wk laying period.

Animals↗

PubMedDate not supplied

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines.

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3' border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk.

Amino Acid Sequence↗

PubMed2006

DAP12 signaling regulates plasmacytoid dendritic cell homeostasis and down-modulates their function during viral infection.

DAP12 is an ITAM-containing adaptor molecule conveying activating properties to surface receptors on many cell types. We show here that DAP12 paradoxically down-modulates plasmacytoid dendritic cell (pDC) cytokine production in vivo during murine CMV (MCMV) infection. Higher levels of IFN-alphabeta and IL-12 were detected upon MCMV infection or CpG treatment in DAP12-deficient (DAP12(o)) mice as compared with wild-type (WT) mice. This resulted from altered homeostasis and enhanced responsiveness of pDCs in DAP12(o) animals. Increased numbers of pDCs were observed in the periphery of both naive and MCMV-infected DAP12(o) mice. A higher proportion of pDCs was activated in infected DAP12(o) mice, as demonstrated by intracellular staining using an optimized protocol for simultaneous detection of IFN-alpha and IFN-beta. The homeostasis of WT and DAP12(o) pDCs did not differ in mixed bone marrow chimeric mice. In addition, a similar efficiency of pDC differentiation was observed in vitro in Fms-like tyrosine kinase receptor 3 ligand cultures of WT and DAP12(o) bone marrow cells. This suggests that DAP12 signaling effects on pDC homeostasis are indirect. In contrast, in response to CpG, DAP12-mediated effects on both IL-12 and IFN-alphabeta production were intrinsic to the pDCs. However, in response to MCMV, only IL-12 but not IFN-alphabeta production was affected by pDC-intrinsic DAP12 signaling. Thus, DAP12 signaling in pDCs can mediate different regulatory effects on their functions, depending on the mechanisms of pDC activation. The potential implications of the regulation of pDC functions by DAP12 for promoting health over disease are discussed.

Adaptor Proteins, Signal Transducing↗