Flow Cytometry

A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quina," the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.

Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.

Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into ohgonucleosomal subumts. Tins type of cell death (apoptosis), which physiologically occurs m the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colonmetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploxd DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4°C) incubation or cyclohexlmide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodmm azide, a substance with cell-kilhng activity through non-apoptotic mechanisms, &d not result in any variation in the normal DNA peak. The flow cytometrlc data showed an excellent correlatton with the results obtained with both electrophoret~c and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptos~s of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes.

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Application of stem cell biology to breast cancer research has been limited by the lack of simple methods for identification and isolation of normal and malignant stem cells. Utilizing in vitro and in vivo experimental systems, we show that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase activity (ALDH) have stem/progenitor properties. These cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential and greatest growth capacity in a xenotransplant model. In breast carcinomas, high ALDH activity identifies the tumorigenic cell fraction, capable of self-renewal and of generating tumors which recapitulate the heterogeneity of the parental tumor. In a series of 577 breast carcinomas, expression of ALDH1 detected by immunostaining correlated with poor prognosis. These findings offer an important new tool for the study of normal and malignant breast stem cells and facilitate the clinical application of stem cell concepts.

Mesenchymal stem cells (MSCs), the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF-Rb expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long-term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.

Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an ␣-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the ␣-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full ␣-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4°C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.

repertoire, there has been considerable interest in determining how much of this expansion represents virusspecific CD8 T cells and how much of it is due to activation of T cells not specific to the immunizing antigen Limiting dilution analysis (LDA) to quantitate virus-spe-Viral infections induce extensive T cell proliferation in cific cytotoxic T lymphocytes (CTL) has shown that only vivo, but the specificity of the majority of the rea small fraction (1%-5% at most) of the activated CD8 sponding T cells has not been defined. To address this T cells are antigen specific at the peak of the primary issue we used tetramers of MHC class I molecules response (Doherty et al., 1992; Ahmed and Gray, 1996; containing viral peptides to directly visualize antigen- . This has led to the hypothesis specific CD8 T cells during acute LCMV infection of that most of the CD8 T cell expansion is not antigen mice. Based on tetramer binding and two sensitive specific and represents bystander activation and/or assays measuring interferon-␥ production at the sincross-reactive stimulation of nonspecific cells. Experigle-cell level, we found that 50%-70% of the activated mental evidence for cytokine-mediated bystander T cell CD8 T cells were LCMV specific (2 ؋ 10 7 virus-specific proliferation during viral infection has come from studies cells/spleen). Following viral clearance, antigen-speby , who showed that type 1 interfercific CD8 T cell numbers dropped to 10 6 per spleen ons can induce proliferation of activated (CD44 hi ) CD8 and were maintained at this level for the life of the T cells and that this proliferation occurs independently mouse. Upon rechallenge with LCMV, there was rapid of signaling through the T cell receptor (TCR). Two recent expansion of memory T cells, but after infection with studies (Ehl et al., 1997; Zarozinski and Welsh, 1997) the heterologous vaccinia virus there was no detecthave questioned the contribution of bystander activaable change in the numbers of LCMV-specific memory tion in viral infection, but the issue remains unresolved CTL. Therefore, much of the CD8 T cell expansion seen because the specificity of the majority of the responding during viral infection represents antigen-specific cells T cells has not been defined. and warrants a revision of our current thinking on the In this study we have reexamined the question of size of the antiviral response.

Recent observations indicate that, in several types of human cancer, only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the ''cancer stem cell'' (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues, either primary tissues collected from surgical specimens or xenografts established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, were disaggregated into single-cell suspensions and analyzed by flow cytometry. Surface markers that displayed intratumor heterogeneous expression among epithelial cancer cells were selected for cell sorting and tumorigenicity experiments. Individual phenotypic cancer cell subsets were purified, and their tumorinitiating properties were investigated by injection in NOD/SCID mice. Our observations indicate that, in six of six human CRC tested, the ability to engraft in vivo in immunodeficient mice was restricted to a minority subpopulation of epithelial cell adhesion molecule (EpCAM) high /CD44 ؉ epithelial cells. Tumors originated from EpCAM high /CD44 ؉ cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Analysis of the surface molecule repertoire of EpCAM high /CD44 ؉ cells led to the identification of CD166 as an additional differentially expressed marker, useful for CSC isolation in three of three CRC tested. These results validate the stem cell working model in human CRC and provide a highly robust surface marker profile for CRC stem cell isolation.

T H -17 cells are a distinct lineage of proinflammatory T helper cells that are essential for autoimmune disease. In mice, commitment to the T H -17 lineage is dependent on transforming growth factor-b and interleukin 6 (IL-6). Here we demonstrate that IL-23 and IL-1b induced the development of human T H -17 cells expressing IL-17A, IL-17F, IL-22, IL-26, interferon-c, the chemokine CCL20 and transcription factor RORct. In situ, T H -17 cells were identified by expression of the IL-23 receptor and the memory T cell marker CD45RO. Psoriatic skin lesions contained IL-23-producing dendritic cells and were enriched in the cytokines produced by human T H -17 cells that promote the production of antimicrobial peptides in human keratinocytes. Our data collectively indicate that human and mouse T H -17 cells require distinct factors during differentiation and that human T H -17 cells may regulate innate immunity in epithelial cells.

Systemic lupus erythematosus (SLE) is a complex, inflammatory autoimmune disease that affects multiple organ systems. We used global gene expression profiling of peripheral blood mononuclear cells to identify distinct patterns of gene expression that distinguish most SLE patients from healthy controls. Strikingly, about half of the patients studied showed dysregulated expression of genes in the IFN pathway. Furthermore, this IFN gene expression ''signature'' served as a marker for more severe disease involving the kidneys, hematopoetic cells, and͞or the central nervous system. These results provide insights into the genetic pathways underlying SLE, and identify a subgroup of patients who may benefit from therapies targeting the IFN pathway.

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The role of tumor-infiltrating immune cells in the early metastatic invasion of colorectal cancer is unknown. methods We studied pathological signs of early metastatic invasion (venous emboli and lymphatic and perineural invasion) in 959 specimens of resected colorectal cancer. The local immune response within the tumor was studied by flow cytometry (39 tumors), lowdensity-array real-time polymerase-chain-reaction assay (75 tumors), and tissue microarrays (415 tumors).

induced the orphan nuclear receptor RORγt, which in synergy with STAT3 promoted IL-17 expression. IL-6 therefore orchestrates a series of 'downstream' cytokine-dependent signaling pathways that, in concert with TGF-β, amplify RORγt-dependent differentiation of T H -17 cells.

Contact between dendritic cells (DC) and resting T cells is essential to initiate a primary immune response. Here, we demonstrate that ICAM-3 expressed by resting T cells is important in this first contact with DC. We discovered that instead of the common ICAM-3 receptors LFA-1 and alphaDbeta2, a novel DC-specific C-type lectin, DC-SIGN, binds ICAM-3 with high affinity. DC-SIGN, which is abundantly expressed by DC both in vitro and in vivo, mediates transient adhesion with T cells. Since antibodies against DC-SIGN inhibit DC-induced proliferation of resting T cells, our findings predict that DC-SIGN enables T cell receptor engagement by stabilization of the DC-T cell contact zone.

The functional properties of dendritic cells (DCs) are strictly dependent on their maturational state. To analyze the influence of the maturational state of DCs on priming and differentiation of T cells, immature CD83 Ϫ and mature CD83 ϩ human DCs were used for stimulation of naive, allogeneic CD4 ϩ T cells. Repetitive stimulation with mature DCs resulted in a strong expansion of alloreactive T cells and the exclusive development of T helper type 1 (Th1) cells. In contrast, after repetitive stimulation with immature DCs the alloreactive T cells showed an irreversibly inhibited proliferation that could not be restored by restimulation with mature DCs or peripheral blood mononuclear cells, or by the addition of interleukin (IL)-2. Only stimulation of T cells with mature DCs resulted in an upregulation of CD154, CD69, and CD70, whereas T cells activated with immature DCs showed an early upregulation of the negative regulator cytotoxic T lymphocyte-associated molecule 4 (CTLA-4). These T cells lost their ability to produce interferon ␥ , IL-2, or IL-4 after several stimulations with immature DCs and differentiated into nonproliferating, IL-10-producing T cells. Furthermore, in coculture experiments these T cells inhibited the antigen-driven proliferation of Th1 cells in a contact-and dose-dependent, but antigen-nonspecific manner. These data show that immature and mature DCs induce different types of T cell responses: inflammatory Th1 cells are induced by mature DCs, and IL-10-producing T cell regulatory 1-like cells by immature DCs.

Conventional chemotherapies are initially effective in controlling tumor growth ( 1 ), yet many patients relapse over time. At least two major explanations exist for these observations. The first is that all cancer cells acquire resistance, resulting in decreased overall sensitivity to therapy over time. In this case, the relative proportion of cells in residual tumors with tumorigenic properties would be expected to be similar before and after treatment. The second explanation is that a rare subpopulation of cells with tumorigenic potential is intrinsically resistant to therapy. In this case, the relative proportion of cells in residual tumors with tumorigenic properties would be expected to increase after treatment. Analogous to the propensity of dandelion roots to regenerate weeds, regrowth of tumors from an intrinsically chemotherapy-resistant subpopulation has been termed the "dandelion hypothesis" ( 2 ). Consistent with this hypothesis, we have previously shown ( 3 ) that the gene expression pattern of residual tumor cells surviving after treatment is

In the bone marrow, the special microenvironment niches nurture a pool of hematopoietic stem cells (HSCs). Many HSCs reside near the vasculature, but the molecular regulatory mechanism of niches for HSC maintenance remains unclear. Here we showed that the induced deletion of CXCR4, a receptor for CXC chemokine ligand (CXCL) 12 in adult mice, resulted in severe reduction of HSC numbers and increased sensitivity to myelotoxic injury, although it did not impair expansion of the more mature progenitors. Most HSCs were found in contact with the cells expressing high amounts of CXCL12, which we have called CXCL12-abundant reticular (CAR) cells. CAR cells surrounded sinusoidal endothelial cells or were located near the endosteum. CXCL12-CXCR4 signaling plays an essential role in maintaining the quiescent HSC pool, and CAR cells appear to be a key component of HSC niches, including both vascular and endosteal niches in adult bone marrow.

Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G 2 /M DNA damage checkpoint in response to ␥-irradiation (IR). CHK1 heterozygosity modestly enhances the tumorigenesis phenotype of WNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.

the generation of specific neuronal cell types from ES Biophysics cells recapitulates normal programs of neurogenesis is Columbia University not clear. New York, New York 10032 Spinal motor neurons (MNs) represent one CNS neu-2 Curis, Inc. ronal subtype for which pathways of neuronal specifica-61 South Moulton Street tion have been defined (Jessell, 2000; Lee and Pfaff, Cambridge, Massachusetts 02138 2001). The generation of MNs appears to involve several developmental steps (Figure 1A). Ectodermal cells acquire an initial rostral neural character through the regu-Summary lation of BMP, FGF, and Wnt signaling (Munoz-Sanjuan and Brivanlou, 2002; Wilson and Edlund, 2001). These Inductive signals and transcription factors involved in rostral neural progenitors acquire a spinal positional motor neuron generation have been identified, raising identity in response to caudalizing signals that include the question of whether these developmental insights retinoic acid (RA) (Durston et al., 1998; Muhr et al., 1999). can be used to direct stem cells to a motor neuron Subsequently, spinal progenitor cells acquire a MN profate. We show that developmentally relevant signaling genitor

Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2+, SH3+, CD29+, CD44+, CD166+, CD13+) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.

Background— Clopidogrel is administered to prevent stent thrombosis; however, the uniformity of platelet inhibition after treatment and the influence of pretreatment reactivity on drug response have not been described. Methods and Results— Platelet aggregation (5 and 20 μmol/L ADP), the activation of glycoprotein IIb/IIIa (PAC-1 antibody), and the expression of P-selectin were measured in patients undergoing elective coronary stenting (n=96) at baseline and at 2 hours, 24 hours, 5 days, and 30 days after stenting. All patients received aspirin (325 mg). Clopidogrel (300 mg) was administered in the catheterization laboratory and followed by 75 mg daily. There was marked interindividual variability in drug response as measured by all markers that showed a normal distribution. Resistance, defined as baseline aggregation (%) minus posttreatment aggregation (%) ≤10% by 5 μmol/L ADP, was present in 31% and 15% of patients at 5 and 30 days, respectively. Patients with the highest pretreatm...

Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNg. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8 + T cells expressing cognate T cell receptors (TCR).

10. The LAGEOS satellites are heavy brass and aluminum satellites, of about 406 kg, completely passive and covered with retroreflectors, orbiting about 6000 km above Earth's surface. LAGEOS, launched in 1976 by NASA, and LAGEOS II, launched by NASA and ASI in 1992, have an essentially identical structure but are in different orbits. The semimajor axis of LAGEOS is a I Ϸ 12,270 km, the period P I Ϸ 3.758 hours, the eccentricity e I Ϸ 0.004, and the inclination I I Ϸ 109.9°. For LAGEOS II, a II Ϸ 12,163 km, e II Ϸ 0.014, and I II Ϸ 52.