MacScan 294 Serial Number [2021]
The channel list in QuickTV is based on the channel names entered in HDHomeRun Setup. If a channel does not have a name entered, it will not show in the list. If no channels have names entered, it will report that a channel scan is required. Simply go into HDHomeRun Setup and make sure all the channels that you want to access have names entered, and if you want, numbers.
MacScan 294 Serial Number
If you are using a Motorola CableCARD, go to the web page for your HDHomeRun device (click on one of the tuner numbers on the Tuners tab in HDHomeRun Setup), then click on CableCARD Menu, then Network Setup. Look for the VCTID, and VCT or VCT Rcvd entries.
Go to the web page for your HDHomeRun device (click on one of the tuner numbers on the Tuners tab in HDHomeRun Setup), then click on CableCARD Menu. Make sure that CableCARD Validation shows success. If it shows none, contact your cable provider, as the card must be validated in order for copy-protected channels to be received.
Card validation is entirely a function of the cable provider sending the correct message to the CableCARD. The most common cause of problems is simply typing in the wrong numbers. Ask the support representatives from your provider to read back the numbers entered in their system, and make sure they match what you see.
If you are using a Motorola CableCARD, both the Host ID and Data numbers must be entered. One or more of the other numbers may also be needed, though it will vary by provider. You may want to refresh the page after they perform their actions and make sure that the Data number remains the same. If it did change, then this indicates that your provider unpaired or reset the CableCARD. This is not necessarily incorrect, but if they do this, you must provide the new Data number before they try to validate again, or it will fail.
Go to the web page for your HDHomeRun device (click on one of the tuner numbers on the Tuners tab in HDHomeRun Setup), then click on CableCARD Menu. If the Card Manufacturer, Card Authentication, and Card Validation lines all show as none, this indicates that the CableCARD is not in a usable state. The most common cause of this would be if the card is not inserted correctly. The card should be inserted with the colored label facing up, and the 68 pin connector going in to the HDHomeRun. It will require a slight amount of force to seat the card in the socket. Approximately half an inch of the CableCARD will remain outside of the HDHomeRun when fully inserted.
In order to further dissect molecular mechanisms involved in the regenerative capacities, working on neuronal cell cultures would be a powerful additional tool. However, despite the enormous success of zebrafish as an in vivo model system, only a few attempts have been reported so far describing the effective culture of primary neuronal cells from embryonic to adult zebrafish6,7,8,9,10. Moreover, the challenging and time-consuming methods currently used for manual dissection of embryonic neural tissues only permit the processing of a limited number of embryos. Furthermore, these do not yet allow the robust establishment of standardised neuronal cultures but rather result in mixed cell cultures6,7,8,9 even when amended with fluorescence-activated cell sorting8. In mammals, enriched neuronal cell cultures can be reliably generated by using magnetic-activated cell sorting (MACS). Since the polysialilated form of the neural cell adhesion molecule (PSA-NCAM) is a distinct marker of immature neuronal-restricted progenitors (NRPs)11,12,13, MACS with microbeads conjugated to an antibody against PSA-NCAM can be used to generate cultures of mammalian NRPs14,15, which subsequently differentiate into neurons but not glial cells11,12,13.
Here we show for the first time the successful application of a MACS based technique in zebrafish. By using a semi-automated dissociation process along with anti-PSA-NCAM microbeads, we isolated immature neuronal cells from a large number of embryonic zebrafish. Our simple, cheap and reproducible method allows the large-scale generation of enriched and viable in vitro cultures of zebrafish NRPs and lays the ground for the establishment of differentiated neuronal cell cultures that will be useful to study neurogenesis or axonal regeneration.
We subsequently characterized the remaining 27% of NF-negative cells. Since we used a culture medium that promotes the survival of neural cells, we hypothesized that the majority of the non-neuronal cells were glial cells. We determined the number of glial cells in the positive fraction by their expression of glial fibrillary acid protein (GFAP; Fig. 3c), an intermediate filament that is expressed in mature astrocytes as well as in neural or glial progenitor cells21. As PSA-NCAM can also be expressed in oligodendrocyte progenitors15,22, we identified these and other glial progenitors by using an antibody against a ganglioside-specific antigen (A2B5; Fig. 3d), a marker for glial-restricted progenitors (GRPs)23 that has already been described in the brain of other teleost fish24,25. The staining revealed that most of the NF negative cells expressed GFAP (17.5 4.0%) and A2B5 (11. 9 2.7%) (Fig. 3e). Taken together, the glial assays suggest that the remaining, non-NF positive cells in our cultures were predominantly PSA-NCAM positive GRPs.
The currently available methods for establishing neuronal cell cultures in zebrafish are insufficient for large-scale experiments. The labour-intensive and time-consuming microdissection of neural tissues only allow the processing of a small number of embryos and the resulting cultures contain only a small fraction of neuronal cells6,7,8,9. We have employed a semi-automated dissociation that allows the simultaneous processing of a large number of zebrafish embryos in less than an hour. Nevertheless, culturing the resulting cell population led to a heterogeneous primary cell culture, similarly as in blastula-derived cell cultures6. We therefore used magnetic-activated cell sorting (MACS) to specifically enrich neuronal cells in zebrafish cell populations. We show that magnetic anti-PSA-NCAM microbeads are applicable for zebrafish and can easily be used to separate neuronal-restricted progenitors (NRPs) for subsequent long-term neuronal cell cultures. Because MACS is comparatively simple and cost-effective to establish, compared e.g. to fluorescent-activated cell sorting (FACS), our methods would be suitable for many labs. If a higher purity is required, our findings suggest that a first major step should be the depletion of PSA-NCAM expressing glial-restricted progenitors (GRPs) by using MACS along with anti-A2B5 microbeads.
Sperm DNA integrity assessment by terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL). (A) Diagram showing staining of sperm DNA for detection of DNA breaks employing the TUNEL assay. This figure is reproduced from reference [70], under the Creative Commons Attribution License. (B) TUNEL assay of spermatozoa analyzed by FCM. Forward light scattering (FSC) vs. SSC dot plots are represented in the top. The gates indicate the selected events for subsequent fluorescein isothiocyanate (FITC) analysis. In the bottom, frequency distribution histograms (number of events vs. FITC fluorescence intensity) of spermatozoa stained with TUNEL are shown. Negative (omitting the TdT enzyme) and positive (spermatozoa treated with DNAse I) controls were employed. The horizontal line (M1) indicates spermatozoa that are positive for the TUNEL technique, and it was adjusted arbitrarily to obtain about 1% TUNEL-positive events in the negative control. The positive control presented 98.48% TUNEL-positive sperm. Two examples of patient samples are shown. Sample from patient 1 presented a high level of TUNEL-positive cells (40.99%), while patient 2 sample exhibited a low proportion of TUNEL-positive cells (9.86%). (This figure is republished with permission of John Wiley and Sons from reference [67]; permission conveyed through Copyright Clearance Center, Inc.; license number 4977690417168).
FCM has also made relevant contributions regarding other medical conditions such as testicular cancer. Testicular germ cell tumors (TGCTs) arise from germ cell neoplasia in situ, originally described as atypical spermatogonia in testicular biopsies of patients that later on developed testicular cancer [71]. These tumors are classified into two main types: seminoma and non-seminomas, the latter characterized by loss of germ cell phenotype and activation of somatic differentiation. The histology of clinical TGCT samples is frequently mixed within a single tumor mass [72]. TGCTs have been shown to have low mutation rate, marked aneuploidy, and universal gain of chromosome arm 12p [73,74,75,76,77]. The aneuploid nature of TGCTs allows for DNA content-based FCM analysis and sorting (see next section) of samples of interest, which provide enriched tumor populations for downstream analyses. Barret and colleagues (2019) [78] recently described a combined approach of DNA content-based FCM, whole genome copy number analysis, and whole exome sequencing, which allowed the interrogation of the genomes of both primary and metastatic tumors, and provided a unique analysis of refractory TGCTs (Figure 6).
Combination of fluorescence activated cell sorting (FACS) with antibody labeling for the isolation of cell types not distinguishable in FCM profiles by sole multi-parametric analysis. Two examples are shown. (A) Isolation of undifferentiated germ cells from neonatal mouse testes, employing Oct4-Gfp transgenic mice and antibody labeling against KIT. A sorting strategy for undifferentiated germ cells (Oct4-GFP+/KIT-) by serial gating is shown. This figure is republished with permission of The Company of Biologists Ltd. from reference [113]; permission conveyed through Copyright Clearance Center, Inc.; license ID 1079949-1. (B) Purification of spermatogonia from adult human testes, employing FCM and antibody labeling. Sorting strategy for cells with the phenotype HLA-ABC-/CD49e-/THY1dim/ITGA6+/EpCAMdim that corresponds to undifferentiated spermatogonia, is shown. Republished with permission of Elsevier from reference [105]; permission conveyed through Copyright Clearance Center, Inc.; license number 4964010165763. In both examples (A,B), the isolated cells were subsequently employed for single-cell RNAseq (scRNAseq).