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  • Sebastian Schmitt added an answer in Amacrine Cells:
    4
    Role of Frmd7 in Rho/GDPase signalling in the retina?

    Hi, I'm trying to investigate the effect of Frmd7 gene (a gene involved in idiopathic nystagmus, an eye condition) in Rho/GDPase signalling, it has been reported that it plays role in activating Rac1 and reorganisation of actin cytoskeleton. I want to look at Rho/GDPase signalling in presence and absence of Frmd7 in the retina, specifically in amacrine cells (where it's expressed). Can I do that with specific markers for Rho/GDPase signalling, and look at changes in activity in wild type and mutant retina lacking Frmd7 (I have a knockout model for Frmd7)?

  • Mirella Ejiugwo added an answer in DPPH:
    15
    Is there a detailed protocol for measuring the antioxidant activity of orange juice and orange using DPPH method?

    Hello All,

    I am presently doing a short period undergraduate project based on assessing the antioxidant activity of orange and orange juice using the DPPH method. However, upon incubating my positive control (different known concentrations of ascorbic acid, ranging from 1 to 10 nanograms per ml) with DPPH reagent, my spectroscopic readings are so inconsistent: they actually make no sense and, thus, cannot yield a useful standard curve. Could there be a detailed protocol to execute this assay successfully please? Thanks in advance.

    Mirella Ejiugwo

    Dear Mr O'Sullivan,

    I wish to plot a graph of % DPPH inhibition versus sample concentration. Looking at the needed formula, I wonder if the absorbance of 0 mM, as seen in your protocol, can be used as control to calculate % RDSA please.

    Earnestly awaiting your response.Thanks.

  • Avichal Rajemahadik asked a question in Protein Refolding:
    New
    Protein refolding and H/D exchange ?

    1)

    I have protein which is studied for H/D exchange at Ph 6 and my instructor told me to perform one more experiment at pH 7.4 and he said that we expect rate to be higher than 25 folds ? How come 25 folds? For each one unit increase in Ph it is 10 fold increase in rate, so it should be 14 fold … Isnt it ?

    2)

    Also how people say that at Ph 5 refolding will happen in 100 msec and at 10 degree (temperature) it is factor of 10 …. How many msec will it take at different pH to refold protein? Also how this number will vary with temperature ?

  • Erkki J. Brändas added an answer in Cognitive Systems:
    99+
    Is Chalmers' so-called "hard problem" in consciousness real?

    In his 2014 book "Consciousness and the Brain: Deciphering How the Brain Codes Our Thoughts" Stanislas Dehaene wrote "Chalmers, a philosopher of the University of Arizona, is famous for introducing a distinction between the easy and the hard problems. The easy problem of consciousness, he argues, consists in explaining the many functions of the brain: how do we recognize a face, a word, or a landscape? How do we extract information form the senses and use it to guide our behavior? How do we generate sentences to describe what we feel?

    “Although all these questions are associated with consciousness,” Chalmers argues, “they all concern the objective mechanisms of the cognitive system, and consequently, we have every reason to expect that continued work in cognitive psychology and neuroscience will answer them. By contrast the hard problem is the “question of how physical processes in the brain give rise to subjective experience … the way things feel for the subject. When we see for example, we experience visual sensations, such as that of vivid blue. Or think of the ineffable sound of a distant oboe, the agony of an intense pain, the sparkle of happiness or the meditative quality of a moment lost in thought … It is these phenomena that poses the real mystery of the mind”."

    Stanislas Dehaene's opinion is "that Chalmers swapped the labels: it is the “easy” problem that is hard, while the “hard” problem just seems hard because it engages ill-defined intuitions. Once our intuition is educated by cognitive neuroscience and computer simulations, Chalmers’ “hard problem” will evaporate".

    Personally, I agree with Stanislas Dehaene's opinion.

    Erkki J. Brändas

    Dear Alfredo,

    I understand that you base your conclusions from a “classical” version of communication. To look for the self-referential being, SELF, I have attempted to realize a lower, more fundamental, level in order to avoid “being as an object” – this is really becoming Heideggerian!

    Communication on the lower level, from genes and cells collaborating on the evolutionary process, becomes “being in the world” (“dasein”, not Hegel and not only “mitsein”).

    This is of course to distinguish between the Self and its projected images, but on “the lower level” this distinction is not significant.

    Hence on the “lower level” also cognition becomes concerned with physical being and not just knowledge.

    Is this what Arnold is saying? It is easier to formulate this in mathematics rather than words.

  • Gitz Negi asked a question in Financial Development:
    New
    What are the drawbacks of using fixed effects method on dynamic panel model?

    Dear Members,

    Could you please mention the drawbacks of using fixed effects method on dynamic panel model in the context of financial development and economic growth.

  • Koushik Majumdar added an answer in Endemic Plants:
    3
    Can any body helps to mentioned any literature mentioning the nativity of the plants?

    Generally in the flora of any region content two major components one is its endemic plants content and the other is the exotic and naturalized floristic elements, besides these there are some some plants which are originated in that region and then it spread to other country.  Dr. D. Chatterjee (1940 and 1960) in his work exhibit the route of migration of different floristic elements from different parts of the world in India.  I am very interesting to know such migration route of the plants from different parts of the world to India and also wants a list of the exotic plants of the Indian subcontinent along with their native country and the time of immigration. 

    Koushik Majumdar

    The biogeographical classification of India has been attempted variously by earlier workers (Hooker 1854, Clarke 1898, Chatterjee 1940, Puri 1960, Rodgers & Panwar I988, Rao 1994). They have unanimously pointed out the enormous variation in vegetation pattern. According to Rodgers and Panwar (1988) the Indian region is divided into  10 biogeographic zones, each with its own unique but distinct natural vegetation representing three basic biomes and two natural realms as identified by Udvardy (1975).

    The flowering plants of India  represent 6% of the world’s known flowering plants (Nayar 1977). However, after critical analysis, subsequent phyto-geographers have convincingly concluded that India has a flora of its own and as many as 33% taxa of Indian flora are endemic to the present political boundaries of India. The total endemic genera represent 6.5%  in India. For understanding the migration route of  plants from different parts of the world to India following valuable publication may help you:

    1. Hajra P K and Rao R R .1990. Distribution of vegetation types in northwest Himalaya with brief remarks on phytogeography and floral resource conservation; Proc. Indian Acad. Sci. 100 263-277.

    2. Jain S K .1986. The grass flora of India - A synoptic account of uses and phytogeography; Bnll. Bot. Snro. India 28 229-240.

    3. Nayar M P. 1977. Changing patterns of Indian Flora; Bull. Bot. Surv. India 19 145-154.

    4. Nayar M P. 1980. Endemism and patterns of distribution of endemic genera (angiosperms); 1'. Econ. Tax. Bot. pp. 199-110.

    5. Rao R R. 1994. Biodiversity in India (Floristic Aspects) pp. 315 (Dehra Dun : Bishen Singh Mahendra Pal Singh).

    6. Rao R R. 1997. Diversity of Indian flora. Proc. Indian Acad. Sci. B 63: 127-138.

    7. Schuster R M. 1976. Plate Tectonics and its bearing on the Geographical Origin and Dispersal of Angiosperms; in Origin and Early Evolution of Angiosperms pp. 48-138 ed C B Beck (London: Columbia Univer. Press).

  • Lawrence Broxmeyer, MD added an answer in Wisdom:
    21
    What is wisdom?

    Philosophic, religious, and opinion responses are acceptable.

    Lawrence Broxmeyer, MD

    "What is wisdom?"

    Hi Mariano. With regard to your interesting question, let me simply quote: "The beginning of wisdom is the fear of the Lord."  -Proverbs 9:10

  • Albert Manfredi added an answer in SDR:
    2
    Can anyone Please suggest How to set a threshold value to sensing a real time signal?

    For sensing a real time signal by using SDR what is the procedure to follow to set the threshold value. In most of the documents its mentioned as trial and error method. Is it the average of the power. But then if noise power is more  or less then it leads to mis-detection  and false alarm. Can any one suggest a way to set the threshold to detect a real time signal using SDR?

    Albert Manfredi

    I think your question is, if you want to determine whether an RF channel is in use, for cognitive radio, what should you use as the signal threshold level? This isn't only related to software defined radio (SDR), but whenever dynamic spectrum sharing is the issue.

    And the answer depends on who is sharing the spectrum.

    For example, let's say you want to use the "TV white spaces." These are TV channels that are presumably not in use, in that location.

    For this, you need to determine first what the sensitivity of typical TV receivers is. This gives you an idea of what a typical TV set would accept as a usable signal. Then you have to accept the possibility that just maybe, the exact spot in which you are trying to sense whether a TV station is present might be blocked from the TV station's signal, to some extent. Possibly, if you move a small distance, you would receive a usable signal.

    To give some reasonable numbers. Typical digital TV sets have a sensitivity of roughly -85 dBm or so, and this measure, along with other measures such as antenna heights and antenna gains, are assumed when calculating reception contours of TV stations. To ensure that a cognitive radio does not interfere with TV signals, within a TV station's legal reception contour, you need to set the threshold reasonably below that. Say, some level less than -100 dBm.

    What makes spectrum sharing in TV bands quite difficult is that TV signals from very far away can be legitimate. Unlike many other examples, such as cellular radio or WiFi, in which far away signals are usually not important. In cell radio or WiFi, usually the signals that matter are the strongest ones. It's perfectly okay for your home WiFi to overpower a detectable but weak signal from several houses down the street.

  • Peter Kapusta added an answer in FRET:
    5
    Would you tell me, how to match the probe to the quencher?

    Dear Community,

    I don't know on what basis people adapts its probe, a simple example: why does Trp quench the iodine, oxygen, caffeine and acrylamide, among others. Why does rhodamine quench the Trp?

    Peter Kapusta

    Agnieszka, the problem is that quenching is a quite general phenomenon. Asking for a specific probe for a certain quencher is an unusual (but interesting and meaningful !) question.

  • Gitz Negi added an answer in Fixed Effect:
    5
    Dear all, is there any difference between 1.) fixed effects model with Initial GDP and 2.) fixed effects model with lagged depndent variable (GDP)?

    Dear All. My research topic is Financial development and economic growth. can you please answer following queries :- 

    1.) is there any difference between a.) fixed effects model with Initial GDP and b.) Fixed effects model with lagged dependent variable (GDP).

    2.) And please do comment on fixed effects with dynamic residual. 

    Best Regards

    Gitz

    Gitz Negi

    @Göksu Aslan, @Muhammad Tariq Majeed, @ Connie Bayudan-Dacuycuy @Avijit Debnath...Thank you very much for your helpful replies. 

  • Sudarson S. Sinha added an answer in Fluorescent Dye:
    7
    What are the principles behind subtracting/normalizing background fluorescence?

    One manufacturer protocol suggests subtracting background fluorescence from the raw fluorescence reads (F - Fo).  Other protocols suggest dividing F/Fo.  Yet another protocol suggests taking (F - Fo)/Fo.  What are the strengths of each approach?  Does anyone know of concise references for this topic?

    Thank you for your help.

  • Pier-Eric Chamberland added an answer in SPSS 22:
    5
    Does anyone know about multiple imputation for categorical variables?

    I have recently collected my data with Likert type questionnaires, and want to impute missing values by using SPSS 22.

    Can anyone suggest me anything about Multiple imputation with that kind of data? Is it OK to run with categorical variables or should I run imputation for scale ones? (for total scores?)

    Thanks in advance

    Seyda  

    Pier-Eric Chamberland

    Dear Seyda,

    I understand that you would like to impute values for a group membership variable.

    The imputation technique can be increasingly complex with the number of categories. For example, you could do imputation on a few binary variables but imputation of nominal multicategorical variables should probably be kept to a minimum. 

    The MICE package in R offers different imputation methods for binary (logreg) and nominal (polyreg and LDA). You can perform different imputation methods for different types variables simultaneously in R, while it is impossible or more imprecise with the missing data module of SPSS.

    I hope that helps. Can you describe your variables?

  • Syed Abbas Jafar added an answer in Fitness:
    99+
    What is your definition of 'fitness'?

    It's all in the question

    Thanks!

    Syed Abbas Jafar

    Marcel et al.:

    You may like to have a look at this link for the history of word "FITNESS" through ages conveying varying shades of meaning.

    https://www.unm.edu/~lkravitz/Article%20folder/history.html

    Best

    Syed

  • Lawrence Broxmeyer, MD added an answer in Risk Assessment:
    36
    What are a chemical company’s top risks and how likely are they to occur?

    Any chemical company's wide risk assessment process should be responsive to change in the business environment. A robust process for identifying and prioritizing the critical enterprise risks, including emerging risks, is vital to an evergreen view of the top risks. Are there any organizational “blind spots” warranting attention in a chemical company?

    Lawrence Broxmeyer, MD

    "What are a chemical company’s top risks and how likely are they to occur?"

    Hi Roland. Unfortunately, this is not always predictable from the onset and might take years, if not decades to determine potential carcinogenic or damaging/disabling systemic effects to the tissue organ-systems of local inhabitants. This is why, as already mentioned, the best immediate defense is a good offense - putting zoning ordinances in play which prevent the construction of such chemical companies in areas of high population density.

  • Feyza Nur Arslan added an answer in Unilamellar Liposomes:
    5
    How to fix unstable GUVs with particles/tubes inside?

    After electroformation, our unilamellar vesicles act very dynamic and unstable on BSA passivated slides (especially as their sizes get bigger). We sometimes observe them bursting and reforming. Also most of the vesicles have tubular structures inside which we want to get rid of. The buffer use inside is glucose solution and outside we have small amounts of salt with very similar osmolarity. I would be happy to hear any suggestions. Thanks!

    Feyza Nur Arslan

    1) I never checked the BSA solution as we are washing it off, but I will try that thanks for the suggestion.

    2) We are using DOPC and very little amount of Rhodamine-DOPE to stain.

    3) Electroformation is taking 4 hours in total, and before that we keep the slide with the lipid film in desiccator.

  • Petros Sountoulides added an answer in Prostate Cancer:
    3
    52 yrs cT2 PCa ,PSA =70 normal bone scan ,no LN in MRI,9/12 CORES +VE ,GS 4+3 is RP THE TREATMENT OF CHOICE?

    52 yrs cT2 PCa ,PSA =70  when repeated = 69 ,normal bone scan ,no LN  & localized disease in MRI,9/12 CORES +VE ,GS 4+3 ,is RP the management of choice?

    Petros Sountoulides

    I think so, RP will provide not only local control as well as staging information from LND. According to findings you may then offer ADT or even chemo upfront if L.N positive. Local control is crucial even in the presence of metastatic disease. 

  • Srabani Majumdar added an answer in Polymerization:
    3
    What is the intrinsic properties of rubbery polymer?

    What is the intrinsic properties of rubbery polymer?

    Srabani Majumdar

    Thank you both. Dear Abdelkader, I am preparing polymeric membrane (glassy/rubbery)  for gas separation. I went through  some journal papers and there was a word 'intrinsic property' . Could u please tell me intrinsic property of glassy/ rubbery polymer in case of gas separation? Thanks in advance. 

  • Debra Sharon Ferdinand added an answer in Online Surveys:
    6
    Has anyone ever had an online survey returned with nil response? If so, what is the best way to report it?

    In the middle of a thesis.

    Have set up an online survey via Google Forms, (link verified through pre-testing with multiple computers and different ISP's.) Now I have sent it to my general population and have had zero response; and the closure date approaches very quickly. I can't extend the closure date either.

    If the totality of the response is zero (assumed) how should this be reported?

    Debra Sharon Ferdinand

    Brain:
    I had a similar incident regarding the use of google sheets to collect data. It turned out that Google upgraded their platform to IE 11 but most if not all of my users were running on IE 9 so they could not open the Google sheet to fill in the data. The latter might be a possible reasons for your respondents not returning or responding to your online survey. It could be browser issue.

    Best regards,

    Debr

  • Fabrice Auzanneau asked a question in Gravitational Waves:
    New
    Do gravitational waves affect their measurement devices?

    I have read the recent paper from Abbott et al about the recent discovery of gravitational waves by the LIGO experience. I'm wondering: if gravitational waves are an evidence of a change in the spacetime structure, they also affect the measurement devices (I mean everything except the arms of the interferometer). Was that taken into account in the data processing?

  • Rafik Karaman added an answer in Macrophage:
    1
    Does anyone has Intestinal macrophage isolation protocol?

    Does anyone have Intestinal macrophage isolation protocol? 

    Regards,

    Ilyas

    Rafik Karaman

    Dear Ilyas,

    The following is the required protocol:

    1-Isolation of Cells from Human Intestinal Tissue
    Immunology > Immune cell isolation > Maintenance and differentiation
    Cell Biology > Cell isolation and culture > Cell isolation
    Cell Biology > Cell movement > Cell migration
    Mammalia > Human > Cell line > Cell-based analysis
    Authors: Heli Uronen-Hansson, Emma Persson, Petra Nilsson and William Agace
    Vol 4, Iss 7, 4/5/2014, 2561 views, 0 Q&A

    The intestinal lamina propria contains a dense network of T cells, dendritic cells (DCs) and macrophages, which play an important role in local innate and adaptive immune responses. We have recently identified distinct subsets of DCs (Persson et al., 2013) and macrophages (Bain et al., 2013) in the human intestine. In addition, we have studied T cells in healthy and diseased intestine. Here, we describe two methods for isolating these cell populations: 1) enzymatic treatment and 2) migration based isolation. The enzymatic method can be used to isolate T cells, DC and macrophages, whereas the migration based ‘walk-out’ protocol is suitable for DC isolation, as these cells migrate out from the tissues.


    Materials and Reagents

    Tissue specimens of small (terminal ileum) and large intestine

    RPMI 1640 (Life Technologies, catalog number: 21875-034)

    Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F7424)

    HEPES (Life Technologies, Gibco®, catalog number: 15630-080)

    Penicillin and Streptomycin (Life Technologies, catalog number: 15140-122)

    HBSS (Life Technologies, catalog number: 14180-046)

    EDTA (Life Technologies, catalog number: AM9261)

    Liberase TM (Roche Diagnostics, catalog number: 05401127001)

    DNase I (Sigma-Aldrich, catalog number: D4263)

    Collagenase 1A (0.2 μm-filtered) (Sigma-Aldrich, catalog number: C9891)

    FACS antibodiesT cells
    CD3-PE-Cy7 (SK7) (BD biosciences)
    Pacific blue (PB)-CD8 (RPA-T8) (BD biosciences)
    Quantum dot (QD) 605-CD4 (S3.5) (Life Technologies, InvitrogenTM)
    LIVE/DEAD® Fixable Near IR Dead Cell Stain Kit (Life Technologies, InvitrogenTM)

    DC/macrophages
    CD3-PE-Cy5 (UCHT1) (eBioscience)
    CD19-PE-Cy5 (HIB19) (eBioscience)
    CD11c-PE-Cy7 (3.9) (eBioscience)
    CD103-PE (Ber-ACT8) or CD103-eFluor647 (B-Ly7) (eBioscience)
    CD14-eFluor450 (61D3) (eBioscience)
    HLA-DR-APCeFluor780 (L43) (eBioscience)
    CD20 PE-Cy5 (2H7) (BioLegend)
    TCRab-PE-Cy5 (IP26) (BioLegend)
    Biotin-or PE-Cy7 CD172a (SE5A5) (BioLegend)
    CD56-PE-Cy5 (Alpha Diagnostic Intl)
    Biotin- or FITC-CD11c (MJ4-27G12) (Miltenyi Biotec)
    CD141-PE (AD5-14H12) (Miltenyi Biotec)
    CD45 V500 (HI30) (BD Biosciences)
    Biotinylated antibodies were detected using streptavidin conjugated to PE-Cy7 (eBiosicence) or QDot605 (Life Technologies, InvitrogenTM).
    Dead cells were excluded from analysis using propidium iodide PI (Life Technologies, Molecular Probes®).

    R10 medium (see Recipes)

    Equipment

    Cell strainer (100 μm) (Thermo Fisher Scientific, catalog number: 22363549)

    50 ml Falcon tube

    Polyester filters cut in 10 x 10 cm squares (mesh count 27 threads/cm, mesh opening 250 μm, thread diameter 120 μm) (Tekniska Precisionsfilter JR AB)

    Petri dish (SARSTEDT AG, catalog number: 82.1473)

    Ultra low attachment 24-well culture plate (Sigma-Aldrich, catalog number: CLS3473)

    Pipette

    Needle/forceps

    Surgical scissor/scalpel

    Beakers with lid (VWR International, catalog number: 216-2694)

    37 °C, 5% CO2 cell culture incubator

    Shaker

    Centrifuge

    Microscope

    Multicolour FACS analyser (BD Lsr II flow cytometer)

    Software

    FlowJo software (Tree Star Inc)
    Procedure

    Enzymatic isolation protocolSurgical specimens of human intestine are collected in R10 medium. The tissue is kept on ice and the isolation procedure started optimally within 1 h.

    Underlying muscular layers and fat are removed with scissors and the remaining tissue is cut into small (< 5 mm) pieces with surgical knives (Figure 1). The tissue is placed into a 50 ml Falcon tube. The maximum amount of tissue in one tube should be approximately 4 g.

    Epithelial cells are removed by incubating tissue fragments in 15 ml of HBSS supplemented with 5% FCS, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and EDTA (2 mM) for 15 min at 37 °C. After the incubation, the tissue is shaken vigorously by hand for 15 sec (foam will appear) and the epithelial cells in suspension are removed by filtering through a nylon filter. The tissue pieces are collected from the filter and placed again in 50 ml Falcon tubes.
    Repeat step A3 for a total of three times.
    Confirm the denuded appearance of the tissue by light microscopy (Figure 2).

    Transfer remaining tissue pieces to a Petri dish. Cut in very fine pieces (< 1 mm) using a needle/forceps and a surgical scissor/scalpel. Look carefully if there are any big pieces left, cut more (Figure 1).
    Transfer the fragments into plastic beakers making sure that all pieces are transferred from the Petri dish.
    Wash the tissue fragments with 20 ml of R10 to remove any remaining EDTA, which may otherwise interfere with enzyme activity in the subsequent step. The pieces will sediment to the bottom of the beaker, remove the R10 with a pipette.
    For isolation of macrophages and DC, the tissue fragments are digested for 2 x 60 min at 37 °C in 10 ml of R10 containing Liberase TM (0.13 Wünsch units/ml) and DNase I (20 IU/ml) with magnetic stirring in plastic beakers with lid. Change the medium and enzymes after the first 60 min and then incubate another 60 min. The cell suspensions are pooled after filtering through 100 µM cell strainers. For T cells, incubate in 20 ml of R10 containing collagenase 1A (1 mg/ml) and DNase I (10 U/ml) with agitation for 60 min. The obtained cell suspension is filtered through 70 µM cell strainer.
    The single cell suspension is washed in 20 ml of R10 and pelleted at 177 x g for 10 min.
    Approximately 4 x 106 T cells, 5,000 CD11c+CD103+ DC and 50,000 CD14+ cells can be isolated from each gram of tissue as calculated by cell sorting (weighed after removal of muscular layer). These numbers are however only an estimate as the donor/preparation variation is high.
    Example of flow cytometry analysis from retrieved cell populations is shown in Figure 3.

    Migration based isolation protocol
    The intestine is collected, cleaned and the epithelial cells removed as in the previous section resulting in tiny pieces (< 1 mm) of denuded tissue.
    Approximately 5 pieces of tissue/well are placed in 2 ml of R10 medium in ultra low attachment 24-well culture plates.
    The tissues are incubated for 18 h at 37 °C in 5% CO2 to allow cells to migrate out of the tissue.
    Place the plate on ice for 30 min in order to detach any plate bound cells. The tissue pieces are discarded and the medium containing migrated cells, is collected from the wells and cells pelleted by centrifugation at 177 x g for 10 min at 4 °C.
    Example of staining of the DC subsets is shown in Figure 4.

    Notes

    Both the enzymatic and migration based protocols can also be applied to small biopsy specimens. In this case, tissue does not need to be further trimmed for removal of fat or size reduction. In addition, the epithelial cell removal step should not include shaking and filtering as the amount of starting material can be very low. Instead, the biopsies are kept in the HBSS/EDTA solution for 45 min at 37 °C with low speed magnetic stirring. After the incubation, discard the medium (and the epithelial cells) and proceed as in step A6. We recommend needles instead of pipette tips for the handling of the tiniest biopsies.

    Collagenase 1A preserves the CD4 epitope on T cells, whereas Liberase cleaves CD4.

    FACS Staining
    Cells were preincubated on ice for 10 min in FACS buffer (PBS/2% FBS) containing 5% mouse serum to prevent unspecific antibody binding.

    Cell staining was performed in 100 μl aliquots for 30 min at 4 °C in the dark, followed by a wash in FACS buffer before being analyzed using an LSR II or FACSAria I and FlowJo software.

    Recipes

    R10 medium
    RPMI 1640 supplemented with:
    10% FBS
    10 mM HEPES
    100 U/ml penicillin
    100 µg/ml streptomycin
    50 μg/ml gentamicin

    References

    Bain, C. C., Scott, C. L., Uronen-Hansson, H., Gudjonsson, S., Jansson, O., Grip, O., Guilliams, M., Malissen, B., Agace, W. W. and Mowat, A. M. (2013).Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunol 6(3): 498-510. 

    Persson, E. K., Uronen-Hansson, H., Semmrich, M., Rivollier, A., Hagerbrand, K., Marsal, J., Gudjonsson, S., Hakansson, U., Reizis, B., Kotarsky, K. and Agace, W. W. (2013). IRF4 transcription-factor-dependent CD103(+)CD11b(+) dendritic cells drive mucosal T helper 17 cell differentiation. Immunity 38(5): 958-969. 

     How to cite this protocol: Uronen-Hansson, H., Persson, E., Nilsson, P. and Agace, W. (2014). Isolation of Cells from Human Intestinal Tissue. Bio-protocol 4(7): e1092. http://www.bio-protocol.org/e1092

     To view the figures and the original publication, please use the following link:

     http://www.bio-protocol.org/e1092

    2-Another source:

    UNIT 7.6B Isolation and Purification of Human Intestinal Macrophages
    Lesley E. Smythies1,
    Larry M. Wahl2,
    Philip D. Smith3
    Published Online: 1 JAN 2006

    DOI: 10.1002/0471142735.im0706bs70

    Abstract
    The gastrointestinal mucosa contained within the lamina propria is the largest reservoir of macrophages in the human body. The isolation and study of this population of cells is important for understanding host defense and the pathogenesis of inflammation in the gastrointestinal mucosa. This unit describes methods that can be used to isolate and purify intestinal macrophages. Sources of intestinal tissue that can be used for this isolation include human subjects undergoing gastrojejunostomy for obesity, organ-transplantation donors, or the noninflamed margin of resected segments of small intestine from subjects undergoing resection for surgically indicated reasons.

    http://onlinelibrary.wiley.com/doi/10.1002/0471142735.im0706bs70/abstract

    Hoping this will be helpful,

    Rafik

  • Justin Andrushko asked a question in Low Frequency Stimulation:
    New
    What are the recommended frequencies (Hz) for a high and low frequency stimulation of the biceps brachii for monitoring peripheral fatigue?

    What are the recommended frequencies (Hz) for a high and low frequency stimulation of the biceps brachii for monitoring peripheral fatigue?References?

    I am currently familiar with high 80Hz/ low 20Hz and high 50Hz/low 10Hz frequencies. What is recommended for the biceps brachii?

  • Peter Gray added an answer in Retinopathy of Prematurity:
    2
    How long does one session of ROP (retinopathy of prematurity) examination take?

    I am currently writing a literature review about sucrose as an intervention during ROP examination. I believe in some NICU’s RetCam is used, in others it is not. There is somewhat conflicting comments in relation to how long these examinations last (1 session). I would appreciate a comment.

    Peter Gray

    I agree with Dr Tripathi. The biggest issues in my experience relate to the difficulties in getting to see the baby who is having other procedures done and also the time for the pupils to dilate. We must remember that these are ill babies whom we wish to screen and that their general well being must take the highest priority.

  • Jan Balaz asked a question in Solid State:
    New
    Is Silicon Drift Detector suitable for detection of energetic electrons ?

    Silicon Drift Detector (SDD) are normally used for detection of X-ray photons. Has anybody used them for detection of energetic electrons? Preliminary response from KETEK manufacturer  http://www.ketek.net/products/vitus-sdd/ indicates that the SDD could be damaged so they would not provide guarantee for this. I am impressed by high energy resolution of SDD in comparison with standard ion-implanted solid state detectors that we normally use. I believe it should work - although with some limitations. Has anybody some experience or theoretical note to this issue ?

  • Peter Kapusta added an answer in High Energy Physics Theory:
    38
    Why nobody see that Higgs boson and gravitational waves are the pure theoretical failures?

    Higgs boson does not interact with gluons which give energy for 99% of proton mass. How the Higgs boson gives mass to the proton, nobody has no clear answer. Why are we closing eyes and tolerating this model which does not have a minimal correspondence with physical existence? Energy and mass are both inherent physical properties of every particle. No particle can give mass to another particle; this is a complete misunderstanding. Higgs boson  does not prove anything, Higgs boson is artificially made flux of energy and nothing more. 

    Energy and mass are both inherent physical properties of every particle. They have the origin in the diminished energy density of quantum vacuum.  A given particle cannot be examined without this diminished energy density of quantum vacuum which determines its energy and mass. The idea that particles exist in an empty space deprived of physical properties is the biggest theoretical failure of physics since its existence. It has led  to the idea of Higgs field and gravitational waves which are both pure theoretical failure. No particle can give mass to another particle; this is a complete misunderstanding. Higgs boson  does not prove anything, Higgs boson is artificially made flux of energy and nothing more. And no wave can transmit gravity. 

    Peter Kapusta

    Sure. Irrelevant in this context.

    Special message to Amrit Sorli: "NATURE" is not an authority, it does not deserve capital letters. Have you ever heard about the "STAP cells" nonsense published by that glamour magazine?

  • Vishvas Patil asked a question in Bricks:
    New
    1) How to model brick infilled panel with shear connectors ? 2) How equivqlent struct will remain in compression under acceleartion loading ?

    who worked in Infilled panel subjected to seismic loading

  • Dick Terwel added an answer in Immunohistology:
    3
    Necrosis Markers for Immunohistological Analysis

    Any suggestions for necrosis markers (antibodies) fo immunohistological assays on cerebellum slices? (Paraffin embedded)

    Dick Terwel

    Annexin V/propidium iodide will not be of much use, since both will stain all cells in histological sections, annexin V/PI is meant for unfixed cells in culture.

    Fluoro-Jade staining is suited for detection of necrotic cells in histological sections. See for instance link below.

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2312368/pdf/JPATH172000470.pdf

    Kind regards,

    Dick

  • Pradip Xavier added an answer in Engines:
    1
    Why aircraft engines use spark plug instead of direct injection system (like modern 2-stroke petrol engines)?

    Why aircraft engines use spark plug instead of direct injection system (like modern 2-stroke petrol engines)? I have heard that nowadays direct injection systems have been developed for gasoline or petrol engine to prevent knocking.

    Pradip Xavier

    Conventional combustion chamber in gas turbines are operating continuously, i.e., liquid fuel and compressed air are supplied to the injection systems (typically swirlers). Aerodynamics of these injection systems is designed to sustain a flame continuously: fuel/air mixing, droplets evaporation, burned gases recirculation for ignition. The spark plug is only used to initiate the combustion (when firing the engine) or in case of high-altitude relight if extinction have ocurred.

    Using direct injection systems would imply a new architecture (piston, valves...),  which may need important modifications (and then certification, and cost issues)

  • José Luis Benítez-Ortega asked a question in Family Therapy:
    New
    Has it been stablished a SOFTA-o cut-off point in order to identify cases or sessions with “good enough” expanded therapeuthic alliance?

    I'm looking for previous research using the System for Observing Family Therapy Alliances (Friedlander et al, 2006) stablishing a cut-off point for SOFTA-o results in order to distinguish cases or sessions with "good enough" from others with "insufficient" expanded therapeuthic alliance.

  • Debra Sharon Ferdinand added an answer in E-Learning:
    12
    What is the fate of e learning in higher education?

    E ;earning is a reality now. What will be the fate of e learning in higher education. will it survive?

    Debra Sharon Ferdinand

    The rapid advancement in technology has made learning beyond the walls of the classroom a common-pace experience in teaching and learning, especially for adult learners (working, studying, family commitments). As such, I can see the fate of e-learning as growing from strength to strength to respond to the flexibility, convenience, and inclusiveness (persons with disabilities embrace e-learning) wants of 21st Century learners and teachers. I am attaching one of my short pieces on flexible learning environments that I hope would useful to your question.

    Many thanks,

    Debra