- Niall Logan added an answer:What is appropriate media for MSCs culture?
I would like to know using Alpha MEM having ascorbic acid is good idea or not for mesenchymal stem cells culture. I generally use mice/rat bone marrow stromal cells for MSCs. using alpha MEM containing ascorbic acid induces differentiation of MSCs. I would be happy to know which media is best for MSCs culture.
We culture human MSCs from bone marrow in MEM Alpha - 10%FBS 1% penicillin streptomycin.
Ascorbic acid is only included if you want to stimulate osteogenic differentiation.Following
- Paul David added an answer:Which marker is best for staining of breast cancer stem cells by immunohistochemistry?
I have been collecting breast tumor tissues from patients and am going to study the expression profiling of stem cell regulatory genes. Also, i would like to do assessment of breast cancer stem cells in tissue section by immunohistochemistry.
but am in little bit of confusion regarding markers. Which marker is best for assessment ? ( ALDH, CD133, CD24, CD44)
For the primary FACS selection, the markers which can be used CD45, ALDH1, and EpCAM.
CD45−/ALDH1+/EpCAM+ or CD45−/ALDH1+/EpCAM−.Following
- Constantine Kaniklidis added an answer:Anybody knows a good review on HP-1 and its role in cell senescence?
I am interested in the role of HP-1 in cellular senescence. We know that HP-1 is involved in the formation of senescence associated heterochromatin foci (SAHF), although it does not bind DNA domain. Nothing is known about its regulation and cellular localization in different cell types. I was wondering if someone is aware of a a good literature review on HP-1 and cellular senescence.
There are a number of important papers1-19 in this fascinating arena - in addition to more general reviews12-21 - so I will briefly outline below some of the more relevant literature, and references within these will allow you to pursue this vital topic further.
Cells from aged humans experience a decline in the levels of the heterochromatin 1 proteins (HP1) which are a component of senescence-associated heterochromatin foci (SAHF)1,2. In this connection, important are the results of the recent University of Rochester genetic study of heterochromatin formation in Drosophila3 that strongly demonstrate that heterochromatin levels - with HP1 being an integral component of heterochromatin and in fact controlling heterochromatin levels4,5, which in turn control rRNA synthesis [Larson, above] - significantly influence and prolong life span, with moderately higher levels of heterochromatin promoting longevity, given that it is confirmed that heterochromatin levels decrease with normal aging, and that heterochromatin formation is essential for silencing rRNA transcription (this transcription promoting protein synthesis in general) via repression of unnecessary rRNA synthesis in order to promote genome stability, and longevity.
And investigators at the Fox Chase Cancer Center have demonstrated that HP1 proteins, prior to incorporation into SAHF, are transiently localized to PML bodies, where they colocalize with the chromatin regulator/histone chaperone HIRA6. More importantly, recruitment of HIRA into PML bodies is one of the earliest steps towards senescence in human cells , and we know that blocking entry of HIRA to promyelocytic (PML) bodies prevents formation of SAHF7, and HP-1 is centrally involved in the formation of ALT-associated PML nuclear bodies (APBs)8 playing a role in telomere-associated senescence.
The Cancer Connection:
Along a parallel line of inquiry, many investigators have also pursued the implications of HP1 and SAHF in the oncology context, leading to the hypothesis that the role of SAHF is to counter DNA-damage response (DDR) in order to force the cell into senescence (as opposed to apoptosis) and suggests that SAHF-associated heterochromatinization might be a barrier to tumorigenesis, as suggested by the fact that several cancer cell lines display elevated HP1 levels9. Therefore, opposing senescence-associated changes in heterochromatin HP1 (and H3K9) occur, consonant with the fact that age is a significant risk factor for cancer10.
Narrowing the Role of SAHF/HP1:
However, against all these views, it is critical to note that recent results11 have marshaled evidence that, unlike the DNA damage response marker γH2AX, SAHF is itself not a common feature of cellular senescence - indeed, that SAHF formation is wholly dispensable, and NOT, as commonly first thought, a universal component of senescence, for multiple forms of cellular senescence - and despite the fact that SAHF formation is shared by diverse cultured cell types under oncogenic stress, it nonetheless appears that SAHF are cell-type-restricted under genotoxin-induced and replicative senescence, and that in consequence, again against widely assumed views, that SAFH does NOT represent a reliable common marker to identify cells undergoing senescence in general (it would appear that only γH2AX-positivity, marking constitutive DNA damage signaling, is a reliable senescence marker), leaving a more narrow purpose for SAHF formation in oncogene-induced senescence.
Where We Stand, and Going Forward
Thus, we have evolved to a more nuanced understanding of the more limited role of SAHF and heterochromatin 1 proteins (HP1) in cellular senescence, although it nonetheless remains true that the heterochromatin process is vital, through elevated heterochromatin levels, in promoting genome stability, and longevity via silencing rRNA transcription and thus the repression of unnecessary rRNA synthesis, and it is clear that we will surely have other studies pursuing the exciting field of how heterochromatinization can be modulated to induced prolongation of lifespan, and - in parallel - suppression of tumorigenesis, thus advancing positivity both against aging and cancer.
- Scaffidi P, Misteli T. Lamin A-dependent nuclear defects in human aging. Science. 2006;312:1059–63.
- Cao K, Capell BC, Erdos MR, Djabali K, Collins FS. A lamin A protein isoform overexpressed in Hutchinson-Gilford progeria syndrome interferes with mitosis in progeria and normal cells. Proc Natl Acad Sci USA. 2007;104:4949–4954.
- Larson K, Yan SJ, Tsurumi A, et al. Heterochromatin formation promotes longevity and represses ribosomal RNA synthesis. PLoS Genet 2012; 8(1):e1002473.
- Grewal SI, Elgin SC (2002) Heterochromatin: new possibilities for the inheritance of structure. Curr Opin Genet Dev 12: 178–187.
- Ebert A, Schotta G, Lein S, Kubicek S, Krauss V, et al. (2004) Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev 18: 2973–2983.
- Zhang R, Poustovoitov MV, Ye X, Santos HA, Chen W, et al. (2005) Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA. Dev Cell 8: 19–30.
- Ye X, Zerlanko B, Zhang R, Somaiah N, Lipinski M, et al. (2007) Definition of pRB- and p53-dependent and independent steps in HIRA/ASF1a-mediated formation of senecence-associated heterochromatin foci. Mol Cell Biol 27: 2452–2465.
- Jiang WQ, Nguyen A, Cao Y, Chang AC, Reddel RR. HP1-mediated formation of alternative lengthening of telomeres-associated PML bodies requires HIRA but not ASF1a. PLoS One 2011; 6(2):e17036.
- Di Micco R, et al. Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer. Nat Cell Biol. 2011;13:292–U244.
- O'Sullivan RJ, Karlseder J. The great unravelling: chromatin as a modulator of the aging process. Trends Biochem Sci 2012; 37(11):466-76.
- Kosar M1, Bartkova J, Hubackova S, Hodny Z, Lukas J, Bartek J. Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16(ink4a). Cell Cycle. 2011 Feb 1;10(3):457-68. Epub 2011 Feb 1.
- Burgess RC, Misteli T, Oberdoerffer P. DNA damage, chromatin, and transcription: the trinity of aging. Curr Opin Cell Biol 2012; 24(6):724-30.
- Krishnan V, Liu B, Zhou Z. 'Relax and Repair' to restrain aging. Aging (Albany NY) 2011; 3(10):943-54.
- Lazarus A, Banerjee KK, Kolthur-Seetharam U. Small changes, big effects: chromatin goes aging. Subcell Biochem 2013; 61:151-76.
- Feser J, Tyler J. Chromatin structure as a mediator of aging. FEBS Lett 2011 Jul 7; 585(13):2041-8.
- O'Sullivan RJ, Karlseder J. The great unravelling: chromatin as a modulator of the aging process. Trends Biochem Sci 2012; 37(11):466-76.
- Chung I, Osterwald S, Deeg KI, Rippe K. PML body meets telomere: the beginning of an ALTernate ending? Nucleus 2012 May-Jun; 3(3):263-75.
- Corpet A, Stucki M. Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells. [Chromosoma 2014 May 27.
- Klement K, Goodarzi AA. DNA double strand break responses and chromatin alterations within the aging cell. Exp Cell Res 2014 Sep 8.
- Tsurumi A, Li WX. Global heterochromatin loss: a unifying theory of aging? Epigenetics 2012; 7(7):680-8.
- Campisi J. Aging, cellular senescence, and cancer. Annu Rev Physiol. 2013;75:685-705. doi: 10.1146/annurev-physiol-030212-183653. Epub 2012 Nov 8.
- Reza Fazlalizadeh added an answer:What anesthetic can I use to bleed rats/mice from the orbital plexus?I am interested in doing retro orbital bleeding of mice/rats for doing FACS analysis of the whole blood. I am looking at a rare population. Right now I am using chloroform to do the same but I am not sure whether chloroform affects the cell numbers. Please tell me if chloroform influences the cell counts or any other parameter in the rodents and also suggest alternate and more reliable anesthetics.
You can find your answer here: http://web.jhu.edu/animalcare/procedures/retro-orbital.htmlA.
Anesthesia is required for retro-orbital bleeding.The following anesthetic agents are recommended for this procedure:
Mice - Ketamine (90 mg/kg) and Xylazine (10 mg/kg), IP
Pentobarbital (50mg/kg), IP
Proparacaine (Ophthetic®) - 1 drop per eye
Isoflurane (Drop Method) - contact vet staff
Rats - Ketamine (75 mg/kg) and Xylazine 10 mg/kg), IP
Pentobarbital (50 mg/kg), IP
Topical anesthetic, alone, may only be used in mice. Rats, who require more restraint due to their size, must be bled under general anesthesia. Note that there is the potential for contamination of the blood sample if topical anesthetic is used.
Use of any other anesthetic agents must be identified in the IACUC application.Following
- Carlo Palesi added an answer:Does chlorhexadine affect the viability of thawed stem cells?
Exploring a new catheter which is coated with chlorhexidine for stem cells infusion.
- Michael ER Olausson added an answer:What is the difference between cell engraftment and repopulation?
What is the difference between cell engraftment and repopulation?
I do not see the difference. If you have a malfunctioning tissue it can either be caused by cells missing due to specific targeted destruction, or a by a general destruction. If you believe that remaining signaling proteins in the ECM could "home" the cells needed, there is really no difference. The "homing" signal comes from the matrix proteins, either called repopulation or engraftment. If we want to differentiate between these entities, I would define engraftment the process of producing more than the original blueprint.Following
- Julie Piccand added an answer:EndoC-βH1 human pancreatic beta cell line.I want to use this cell line as a T2DM model. Who are the suppliers of this cell line in India?Following
- Bhagat Patlolla added an answer:Is it more efficient to induce the normal cells directly to the desired cells or induce them first to pluripotent then to desired cells?
These days some normal cells like fibroblast can also be transformed to desired cells type like neuron or cardiac myocytes or blood cell progenitors or other cells instead of inducing pluripotent stem cells then inducing them to differentiate to these cells. The question arises, why do we need to use stem cells rather than use direct transformation techniques. The efficiency and other things can be improved later. Please add your comments! Thanks!
I agree with you all. Tho Direct Differentiation is an attractive method for generating the target cells of interest, currently this technique is limited only to a few cell types. The challenge tho is getting more number of target cells(efficiency) and avoiding Dedifferentiation and Transdifferentiation of the target cells. Also, most importantly, the method of differentiation (non-viral to be precise).Following
- Soeren Turan added an answer:Will human stem cells grow alone (single cell clones in 96well)?
What considerations should I take to ensure a colony will grow from a single plated cell. i.e. should I add supernatent from a confulent colony and do I need to co-culture with other cells? Is there a specific media reagent that will promote growth in an isolated environment? Thanks.
Hi Sergey, is there any easier way to create the dialyzed albumin/medium solution ?
Why is albumin so important? Which albumin should we order (cat no?)
when i plate a 96 well plate with matrigel and rock, how many single clones can i expect, how many can i expect with laminin521?
- Holger Fey added an answer:How can I separate mononuclear cells from cord blood apart from ficoll hypaque?
There are so many papers stating that ficoll hypaque gradient can separate mononuclear cells from cord blood but is there any method apart from this? Kindly share detailed protocol.
Hmm, why would you want to use something different than a Ficoll density gradient to enrich MNCs? The method has been around for ages and there must be tens of thousands of labs that use it routinely. I've used it and it works every time. Take your cord blood and dilute it with an equal volume of 1 x PBS. Then layer the blood on 12 ml of Ficoll in a 50 ml conical tube and spin w/o brake at 2200 to 2400 rpm (depending on the centrifuge and rotor dimensions) for 25 minutes. You could use Percoll instead, but it's more or less the same thing, just a differnt compound.Following
- Younes BOUALLEGUI added an answer:What drives to predict the signaling cascade in response of a particular stimuli?I just want to know how to predict the response of a cell that activated a signalling event. I wish to discover any fundamental or computational tool to predict the signalling events.
If any exist, by giving the search phrase to the web to predict the possible mechanisms at dependent experimental conditions?
Best regards to thomas, realy he didn't let anything to say, very good sayed, i absolutely agree with him.Following
- Mandana Daneshvar added an answer:Does anyone have experience with stem cell growing?
I got embryonic stem cells 1 month ago and cultured them according to the protocol from its company. After 3 X making them passage, they start growing very slowly and not confluent well as we always expect. Everything is optimized and it shouldn't be existed any problem but this time I face to the problem. Does anyone know how I can overcome to the problem or have any suggestion to improve cells growing. I should mention that I use cell medium from company for seeding and growing the cells as well which needs to keep pluripotency of cells . I would be appreciated if anyone give me a clue to overcome the problem.
Thank you for all useful tips that you gave me. Now, It's working out and the cells start growing well. It supposed that it was caused by supplement from company because we used first seeding cells on supplement from company and then changing in differentiated medium for transplantation. Supplemets from company doesn't include many feeders for keeping pluripotency that's why they were growing slowly and I was worried to decline cell number on plate and also growing time but solved it now.Following
- Venil N Sumantran added an answer:How do I reduce cell proliferation while performing wound healing assay?Note: I tried low serum, however, my cells clump under low serum condition. Is there any other way?
Jiang, I have not done this assay myself. So, its better if you get protocol by reading literature.Following
- Joe Graymer added an answer:Aneuploidy in cancer "stem cells"?I have a question concerning aneuploidy in cancer "stem cells".
In most tumors one could observe cells unevenly divide their genetic material and producing aneuploid cells but in most "normal" cells aneuploidy is lethal. I know that there are some mutations allowing cancer cells to survive mild changes in chromosome composition but I believe that aneuploidy that goes too far must be lethal to cancer cells as well. So there should be some kind of balance - mutations increasing fitness but not to much. (so I believe most cases of hugely uneven genetic material composition that we can observe in microscopic analysis of tumors is only a by-product and these cells die just after division).
But how it is in case of cancer "stem cells"? Are they similarly aneuploidic as other "somatic" cancer cells or keep their chromosomes more under control?
You may like this:
- Sabine Siegemund added an answer:Phosflow technique to study signaling in hematopoietic stem cells?I want to study intracellular signaling in mouse hematopoietic stem/progenitor cells. Due to the fact that stem/progenitor cells are a very small population, it would be cool to use phosflow (detection by FACS) - especially the combination with surface marker staining would be very interesting for me ! Does anybody have good protocols - especially for hematopoietic cells?Following
- David Ebertz added an answer:Can anyone help with mammosphere quantification?I'm having trouble quantifying the weekly results of my mammosphere protocol for measuring stemness in cancer cell lines. Using StemCell's protocol, on the day of counting I:
harvest the mammosphere + media
Resuspend in 500uL of mmaosphere media
Transfer 50uL to a 96 well and count the numbers in this dilution
The main problem is that when I get to the 96 well, I've been seeing quite a reduction in the size of my mammospheres. I'm worried that the slight manipulation of the spheres leading up to this are breaking them up. But its also impossible to count if I were to keep the spheres in their normal 6-wells.
Does anyone have any experience or know people who have somewhat mastered mammospheres and could help me figure out how to better quantify? I could really use some help.
So I tried coating my own plate with (mammosphere media + 1.5% agarose). I used 70uL per well of a 96 well plate. I let it dry over night for good measure before seeding. The next day, I seeded cells for mammospher formation with 100uL of media per well. However, the very next day, all the cells were in the center of the dish. It was as they all moved towards the center even though I had laid down the agar mixture.
Has any of you had this problem before? Do I need to use more than 70uL to coat? Is it the wrong % of agar?
- Agnes Forsthuber added an answer:What are the chances that CMV promoter-lead genes in a lentivirus vector will undergo silencing after infecting stem cells?There are couple papers showing CMV is not an optimal promoter for creating stable cell lines from stem cells since this promoter and associated genes have high rate of silencing after transfection/transduction. My goal is to create a lentiviral clone with a non-CMV promoter to transfect stem cells. I have my gene in the gateway entry vector with CMV promoter and Life technologies do not have a lentiviral destination vector with EF1alpha or PGK promoter.
Can I easily excise the gene from the gateway entry vector and reclone it in other lentiviral vectors (non-gateway) with any promoter of my choice?
besides expression in stem cells: I am facing a similar problem in murin melanoma cells.
I successfully transfected murine melanoma cells (B16) with a lentiviral plasmid 5´LTR-CMV-GOI-SV40-ReportergeneRFP/Selectionmarker-3´LTR.
GOI - coding region of my GOI has 350bp and an n-terminal signal sequence and should be secreted.
I find reporter gene expression but no expression of GOI!
Could you maybe share a link of those ublications where the problems with CMV promotor are discussed?
I case of murine melanoma cells-could you suggest any other strong promotor that is consitituively acitve?Following
- Mohammad Ali Sabbaghi added an answer:What do you prefer to use for understanding the migration of cells in vivo after transplantation?Other than GFP, are DAPI or Hoecst suitable for this? My aim is labeling the mesenchymal stem cells and, after 60 days, observing them from the tissue of recepient rat.
Sorry for late answer.
we used DiI in our study and it remained till 90 days post transplantation. As we had limitation for using GFP, DiI was the best option.Following
- Olivia Zabel added an answer:Which transfection reagent is best with reporter assays on stem cells?Which one is enough for maximum efficiency and min toxicity (with min price) when using ready-for-reverse-transfection 96 well plates with mesenchymal stem cells;
Lipofectamine LTX or Lipofectamine 2000 or any other reagent?
PS: I'll use Qiagen's 10 pathway reporter assays along with Promega's Dual-glo luciferase assay system with a number of cell types including primary adipocytes and adipose derived MSCs.
for siRNA/miRNA transfection of MSC I advise a new reagent called Viromer BLUE which was developd by a company called Lipocalyx:
By using Viromer BLUE you will get up to 90% efficiency.
If you have further questions please feel free to contact me: Olivia.Zabel@lipocalyx.de
- Karl Wahlin added an answer:Can anyone help with neuron attachment on glass coverslips?Been differentiating ES/IPS cells into neurons. I need to culture them on glass coverslips for downstream microscopy. I find that neurons don't attach well to coverslips coated with Poly-Lysine, PL/Laminin and PL/LM/Fibronectin combinations. LM alone, coating looks OK except that the attachment is not strong! So, whenever I change medium invariably some cells come off the surface and by the end of incubation period over a couple of weeks many of the cells are gone. Any ideas to get a more firm attachment of neurons to glass coverslips?
1. I would first make sure that the glass has been cleansed of any lipids left over from manufacturing.
2. Acid etch overnight in HCl or Nitric acid (be careful if going the nitric acid route).
3. Wash ~ 10x with lots of water to remove any acids.
4. Coat with poly-ornithine (in Boric acid) at a concentration several fold higher than what you would do for plastics. * this part is very important. * Glass just needs a little more help in getting things to stick.
5. After coating with PolyOrn rinse 3x with water and you should be good to go. A brief period in 10% FCS prior to plating cells helps a lot but isn't critical.Following
- David Rhoads added an answer:Is there a consistent moderate-throughput transient transfection technique for promoter analyses in mouse ES cells?Currently, the ES cells are maintained on a MEF feeder layer but seeded onto gelatin alone in a 48-well cluster for transfection with Lipofectamine 2000. Electroporation or nucleofection would be too cumbersome. I haven't tested other transfection reagents yet or whether including MEFs for seeding would produce an unacceptable background.
Alas, our promoters showed very little activity in the ES cells (as opposed to their activity in HEK-293 cells). Thus, it's more a matter of target cell than transfection path.
Thanks! - DaveFollowing
- Patricia Wilson asked a question:Has anyone used SNL feeders for rat iPSC derivation?
Used as source of Lif?
Great discussion board!Following
- Saleh Alkarim added an answer:Does anyone know of a protocol to differentiate hESC and hiPSC into endothelial cells?
We have been optimizing our protocol for directed cardiac differentiation of human embryoninc stem cells and induced pluripotent stem cells. This protocol (as proposed by Lian et al. in Nature Protocols 2013) now works well in our lab. My specific question therefore is whether anyone has any suggestions to differentiate stem cells towards endothelial cells in a similar way as the Lian protocol works for directed cardiac differentiation (i.e. monolayer based amd growth factor mediated)? Any and all suggestions are welcome. That you very much in advance.
best wishes for yourFollowing
- Nur Afiqah added an answer:Any advice on care of Mesenchymal Stem Cells (MSCs)?I've been working with some bone marrow derived MSCs I got from Texas A&M and I've wondered what other's experience has been with bone marrow derived MSCs (or MSCs from adipose tissue, hUCB etc. doesn't matter). In my experience they tend to be slow growers (plated at ~2000 cells/cm^2 it'll take ~10 days to reach 80% confluency) and senesce quickly (I get like 7-10 passages out of them). I change the media weekly and don't use a pH indicator. I've been using stemlife MSC medium for undifferentiated expansion. This media is interesting to me because I assume its probably pretty basic but it contains what they call "life-factors" which is proprietary of course.
Has anyone on here created their own media or found a particular method of culturing to be the most robust and productive when working with bone marrow derived MSC's?
Dear Purva Sanganeria
As you mentioned that you did not add penstrep in your culture media, so how you prevent/minimise contamination?Following
- Dmitry Klokov added an answer:Can anyone offer suggestions for an automated software for morphological evaluation of Images with some cell clusters from 24 well plate?Eg.:area, perimeter, diameter.We have recently purchased and started using a ImageXpress Micro automated cell imaging system from Molecular Devices. It comes with a software called MetaXpress that is capable of a great variety of measurements and analyses, including morphological ones. I have worked with Image J, Northern Eclipse, some other software packages for the analyses of microscopic images, and none of them compares with MetaXpress for speed and flexibility. Big question for you will be whether it can work with data collected outside of its "mother" hardware imager. You could probably find this info on their web page or contact tech support. Obviously, it is not freeware unlike the one suggested in the post above. Just sharing my experience.Following
- Nand kishor Roy added an answer:Can I use a normal genomic DNA isolation kit for isolation of apoptotic DNA of cancer cell?We have MN kit in our lab nucleo spin, but I want to know whether it can be used for isolation of apoptotic DNA.thank you Pradyumna Mishra Sir for your kind reply.Following
- Andrew S Brack added an answer:Is there evidence for non-satellite cell resident muscle progenitors?On ResearchGate, there has been a very collegial discussion about the role of satellite cells during muscle hypertrophy. A number of us also cited increasing evidence that non-satellite cells play a role as niche support cells and/or can directly participate in the myogenic lineage. So, hoping for a fruitful discussion, lets start to put this data together to see if it forms a coherent picture? Jump in.I'm trying to get an appreciation of the potency of non-satellite cells to make satellite cells (as defined by Pax7). Michael has highlighted the Guttridge paper (beautiful work) as it relates to Cancer cachexia. This is very compelling in my opinion. I'm still left wondering how often this mechanism is activated. How rare are these events in the broader context on satellite cell maintenance and injury response?Following
- Holger Fey added an answer:Which company sells CD105 antibody (anti-rat) conjugated FITC or PE?I cann't find a company that sells a conjugated antibody of CD105. Does anybody have a suggestion? Thanks.I never had to. Luckily whatever antigen I've needed a direct antibody-conjugate for in the past, it was always available, usually from multiple sources (BioLegend, eBioscience, BD Biosciences). Sorry, can't help you regarding the Millipore mAb. I just linked to that one, b/c it says in the description that the mAb is expected to also react with rat endoglin.Following
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