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No, it is not necessary to remove the plasma layer. The protocol has been optimized for maximum PBMC recovery. If you have any more questions, feel free to email us at techsupport@stemcell.com.

Yeah until it cures complex genetic diseases and allows human civilization to become independent in a way that shows we were the “satanic” ones all along.

Hi, it should be possible to apply the protocol to individual brain regions for downstream microglia isolation but we have not tested it. It may require some optimization in terms of the amount of digestion medium per starting tissue, since it is currently optimized as 1 mL of digestion medium per whole brain of an adult mouse (as per the Product Information Sheet for Catalog #18970 EasySep™ Mouse CD11b Positive Selection Kit II: cdn.stemcell.com/media/files/pis/10000003693-PIS_02.pdf). If you have any more questions, feel free to email us at techsupport@stemcell.com.

Absolutely, good lab hygiene practices are crucial. It's also critical to monitor cell quality, including assaying for contamination, throughout the course of experiments. For more information, check out our PSC Cell Quality Hub (www.stemcell.com/psc-cell-quality) or the ISSCR Standards (www.stemcell.com/psc-cell-quality/isscr-standards).

Ficoll™ and other density gradient media act as a layer to separate cell types based on their density. During centrifugation, cells more dense than Ficoll™ (such as red blood cells and granulocytes) migrate below the Ficoll™ layer and pellet at the bottom of the tube. Lymphocytes, monocytes and platelets are not dense enough to penetrate the Ficoll™ layer. These cells therefore collect as a concentrated band at the interface between the original blood sample and the Ficoll™. If the blood sample is mixed with the density gradient medium before centrifugation, this density-based separation cannot happen. If you have any more questions, feel free to email us at techsupport@stemcell.com.

The process of washing the enriched PBMCs twice is performed to remove any residual density gradient medium (and optionally, platelets). If these wash steps are omitted the remaining density gradient medium could have toxic effects on the isolated cells. If you have any more questions, feel free to email us at techsupport@stemcell.com.

Hi, the full protocol can be found here (cdn.stemcell.com/media/files/manual/MADX23032-Maintenance_of_Human_Pluripotent_Stem_Cells_in_mTeSR_Plus.pdf). Specifically, steps on how to coat the plate with Matrigel is detailed in section 4.2.1. You will need to first make aliquots of Matrigel based on the recommended aliquot size/dilution factor listed in the Certificate of Analysis, then load the recommended volume listed in table 1 (section 4.2) into the cultureware. You can also refer to the written version of the video protocol, found here (www.stemcell.com/transitioning-pscs-to-mtesr-plus.html). If you have any more questions, feel free to email us at techsupport@stemcell.com

@@STEMCELLTechnologies Thank you.

A buffy coat contains leukocytes in a concentrated suspension, originating from whole blood or bone marrow. After centrifugation, we remove the concentrated leukocyte band (this is the buffy coat), plus a small portion of the plasma and concentrated red blood cells (RBCs). The target is to concentrate the leukocytes approximately 5-fold while maintaining an equivalent ratio of leukocytes to RBCs (e.g. collect 2 mL of buffy coat when starting with 10 mL of whole blood). You can also refer to our technical tip “How to Prepare a Buffy Coat from Whole Blood” (www.stemcell.com/how-to-prepare-a-buffy-coat.html) for more information. If you have any more questions, feel free to email us at techsupport@stemcell.com.

Hi Marcos, For the wash steps, centrifuging at 300 x g for 8 minutes at room temperature, with the brake on, is recommended. The volume of density gradient medium to add to the SepMate™ tube depends on the size of the tube (15 mL or 50 mL) and the blood sample volume. 15 mL is the volume required for the 50 mL tubes. This information is available in Table 1 of the product information sheet (cdn.stemcell.com/media/files/pis/10000003788-PIS_09.pdf). The volume of density gradient medium has been optimized based on the position of the insert and using a different volume than recommended may affect the performance of the isolation. SepMate™-15 (cat #85420 www.stemcell.com/sepmate-15-ivd.html) is designed to process from 0.5 to 5 mL of whole blood (before dilution), and SepMate™-50 (cat #85450 www.stemcell.com/sepmate-50-ivd.html) is designed to process from 5 to 17 mL of whole blood (before dilution). This information is available in Table 1 of the product information sheet (cdn.stemcell.com/media/files/pis/10000003788-PIS_09.pdf). If you have any more questions, feel free to email us at techsupport@stemcell.com.

Hi, you can learn more about EasySep™, and how to purchase the kit and magnet here: www.stemcell.com/products/product-types/cell-isolation-technologies.html. If you have any questions, feel free to use our LiveChat to get connected with a representative or contact us at techsupport@stemcell.com.

Permanent magnets are used in all of our EasySep™ Magnet systems, so they are suitable for indefinite repeated use as long as there are no signs of damage or wear. If you have any more questions, feel free to email us at techsupport@stemcell.com.

The number of samples that can be processed varies with each kit, and also depends on the sample type and volume. If you are searching for the processing capacity for a particular EasySep™ kit, this information can be found on the Product Information Sheet, which is located in the "Protocols and Documentation" section on the product specific page of our website. If you have any more questions, feel free to email us at techsupport@stemcell.com.

The incubator in the video is not waterless. We do not recommend a specific incubator type or model for the protocol. Any incubator with humidity and gas control to maintain 37°C and 95% humidity in an atmosphere of 5% CO2 in air is suitable. If you have any more questions, feel free to email us at techsupport@stemcell.com.

The tool used in this video at 1:21 is the plunger of a sterile disposable syringe. If you have any more questions, feel free to email us at techsupport@stemcell.com.

Indeed, ReproRNA™-OKSGM encodes the Yamanaka factors to reprogram somatic cells to pluripotent stem cells. This is a non-viral method. The vector will persist in cell culture for as long as B18R treatment is maintained. B18R down-regulates the cells innate response against exogenous RNA. Yoshioka et al., 2013 (pubmed.ncbi.nlm.nih.gov/23910086/) assessed RNA content of cells after the removal of puromycin and B18R selection. They observed that all traces of RNA had cleared by the 8th passage (most had cleared by the 5th). This group failed to detect any integration events in their cell lines, indicating the safety of this system for wild-type genotype iPSC generation. When compared to Sendai virus systems which have been shown to persist for over 11 passages, 5-8 passages are very attractive time savings (see Schlaeger et al., 2015, which also discusses other reprogramming methods:pubmed.ncbi.nlm.nih.gov/25437882/). We have not tested light-controlled reprogramming in house, but optogenetic-controlled cell reprogramming systems are possible in general, see e.g. Wang et al., 2013 (pubmed.ncbi.nlm.nih.gov/36507552/). If you have any more questions, feel free to email us at techsupport@stemcell.com.

@@STEMCELLTechnologies Thanks so much for your answer and more thanks for web-links

Unfortunately this doesnt work so simple. Yes, is it possible to get IPSC, but as "raw substance" they are useless. To make new tissue or whole you need to reprogram (to different) them back to somatic cells. And besides there are atleast two big problems. First - short telomeres, wich you cant make longe without cancer. And second - IPCS have features from their "parents" - IPCS from skin will have feauters of skin cells; and IPCS from muscles will have feauters of muscles.

To mark differentiated areas, we used a Nikon Microscope Object Marker (part # MBW10020). If you have any more questions, feel free to email us at techsupport@stemcell.com.

Hi, this video follows the protocol for isolation with #18063 with the Easy 50 (#18002) magnet that starts on page 5 . Please follow the instructions in the greyed-out column ( cdn.stemcell.com/media/files/pis/10000000743-PIS_03.pdf ). If you have any more questions or would like further clarification on the protocol, feel free to email us at techsupport@stemcell.com.

Each time you solve a logical problem you create new brain connections,.. same when you complete a physical challenge for your own achievement,..

We follow aseptic technique while in the lab even if it is not shown in the video. If you are worried about contamination, you may spray a paper towel with ethanol and wipe the bottom of the flask. If you have any more questions, feel free to email us at techsupport@stemcell.com.

@@STEMCELLTechnologies thank you .

Hi, we would need more clarification. Please email us at techsupport@stemcell.com on how you're hoping to use this for your application.

You can use a standard CO2 incubator designed for tissue culture work. If you have any more questions, feel free to email us at techsupport@stemcell.com.

There is variation in the total number of nucleated cells in the mouse spleen. In general, the older the mouse, the lower the splenocyte yield is. However, the most frequently observed splenocyte yield per spleen is 5 x 10^7 to 1.5 x 10^8 cells. If you have any more questions, feel free to email us at techsupport@stemcell.com.

After the original publication on human cell reprogramming by the Yamanaka lab, Takahashi et al., 2007 (www.cell.com/cell/fulltext/S0092-8674(07)01471-7), many different vector types and combinations of reprogramming factors were used and published. So while reprogramming of adult human cells to iPS cells is in principle all based on the original work done by Yamanaka, you will find many variants of methods by now. One improvement over the original retroviral method was using non-integrating vectors to avoid insertional mutagenesis. The system presented in this video, ReproRNA™-OKSGM, is for example a single-stranded RNA replicon vector that contains five reprogramming factors: OCT4, KLF-4, SOX2, GLIS1, and c-MYC, as well as a puromycin-resistance gene. This RNA vector reprograms somatic cells, such as fibroblasts, into induced pluripotent stem (iPS) cells - similarly to the original approach by the Yamanaka lab. However, while the original paper from Takahashi et al., 2007 (www.cell.com/cell/fulltext/S0092-8674(07)01471-7) used retroviral vectors for the insertion of the reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc into the genome, ReproRNA™-OKSGM contains an additional factor, GLIS1, contains a puromycin selection cassette and is not integrating into the genome. If you have any more questions, feel free to email us at techsupport@stemcell.com.

@@STEMCELLTechnologies thank you so much. I am only a student now so thanks for your detailed explanation.

@@STEMCELLTechnologies Sorry to bother you, but can I ask you one more question? How exactly does a cell react to the appearance of such reprogramming factors within itself? Is it simply important that all factors be present simultaneously, as if we had a logical “0” for the absence of factors and a logical “1” for their presence? Or, for example, is it important that the factors be present in a certain number?

@@kerbalfly529introducing simultaneously will do it most likely

This product is designed to maximize the recovery of dendritic cells from mouse spleen when combined with EasySep™ cell separation technology (e.g.EasySep™ Mouse Pan-DC Enrichment Kit, Catalog #19763 and EasySep™ Mouse Pan-DC Enrichment Kit II, Catalog #19863). For isolation of macrophages, mechanical dissociation is recommended followed by the use of the EasySep™ Mouse F4/80 Positive Selection Kit (Catalog #100-0659). This Tech Tip (www.stemcell.com/technical-resources/methods-library/cell-separation/sample-preparation-and-storage/preparation-of-single-cell-suspensions-from-tissue-or-cell-culture-samples/prepare-single-cell-suspension-from-mouse-spleen.html) might be useful to you. If you have any more questions, feel free to email us at techsupport@stemcell.com.

Hi, there is no significant difference between PBS and DPBS. Both of them contain sodium phosphate, sodium chloride, and, when required, potassium phosphate and potassium chloride. Other preparations, such as DPBS, may or may not contain calcium and magnesium. PBS and DPBS have numerous applications because they are not noxious to cells. Please make sure however that you are using a formulation without calcium and magnesium when generating cell aggregates and passaging hPSC in mTeSR™ Plus. If you have any more questions, feel free to email us at techsupport@stemcell.com.

Hi, this video outlines an enzymatic method to generate a single-cell suspension from mouse spleen prior to the downstream isolation of dendritic cells.

Platelets, by nature, are very sticky, and when activated, will adhere easily to other sticky cell types (i.e. monocytes, eosinophils, etc.), creating cell clumps. The video is describing a method to remove platelets from the enriched mononuclear cells by performing one of the washes at 120 x g for 10 minutes at room temperature, with the brake off. This centrifugation will pellet mononuclear cells but not platelets, so they can be removed by aspirating the supernatant following the spin. Another method to reduce platelet contamination with SepMate™ is to pipette off some of the supernatant above the mononuclear cell layer before pouring to collect the mononuclear cells. Both methods are described in the Product Information Sheet (cdn.stemcell.com/media/files/pis/10000003788-PIS_09.pdf ) for SepMate™. If you have any more questions, feel free to email us at techsupport@stemcell.com.

Thank you and we'll share this feedback with our team.

The protocol uses EasySep™ Mouse CD11b Positive Selection Kit II (www.stemcell.com/easysep-mouse-cd11b-positive-selection-kit-ii.html ). The brain tissue preparation protocol is found in the 18970 PIS (cdn.stemcell.com/media/files/pis/10000003693-PIS_01.pdf ) but it was optimized for adult (>6 weeks) mouse brain samples. Unfortunately, we do not have extensive experience working with postnatal samples for this particular protocol. If you have any more questions, feel free to email us at techsupport@stemcell.com.

@@STEMCELLTechnologies The link is broken. Has it been combined into the NeuroCult Adult CNS kit?

Hi, please try the link again.

Thank you for your suggestion.

Adherent hPSC 2D cultures grown on Corning® Matrigel® with mTeSR™1, mTeSR™ Plus, TeSR™-AOF or TeSR™-E8™ can be transitioned directly into dynamic suspension culture. To successfully expand cells and maintain pluripotency in a suspension culture system, it is crucial to begin with high-quality hPSC cultures. Typically, hPSCs are maintained in 2D on Corning® Matrigel® with mTeSR™1, mTeSR™ Plus, TeSR™-AOF, or TeSR™-E8™, and are passaged with Gentle Cell Dissociation Reagent (Catalog #100-0485) as clumps that are 50 - 200 µm in diameter. For 3D cultures, clumps from 2D cultures are first cultured in 6-well plates on a shaker, then in bottles, and can then be upscaled further to the PBS Mini. For more detailed information, please read this technical tip: www.stemcell.com/robust-scale-up-of-human-pluripotent-stem-cells-using-tesr-3d-media.html. If you have any other questions, feel free to email us at techsupport@stemcell.com.

Yes, you can use Ficoll™, Lymphoprep™, Histopaque®-1077 or any other density gradient medium intended for isolating mononuclear cells with a density of 1.077g/mL. If you have any more questions, feel free to email us at techsupport@stemcell.com.