STEMCELL Technologies is a Canadian biotechnology company that develops specialized cell culture media, cell isolation products, instruments and accessory reagents for life science research. Driven by science and a passion for quality, STEMCELL delivers over 2500 products to more than 80 countries worldwide.
STEMCELL Technologies provides specialized cell culture media for pluripotent stem cell, hematopoietic, mesenchymal, neural, mammary and epithelial research. A full line of cell separation products, including RoboSep™, the fully automated cell separator, is available for the isolation of any cell type from virtually any species. Other products and services include primary human cells, sera, cytokines, antibodies, training courses, Proficiency Testing, and Contract Services.
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Do I need the entire process to be carried out in a biosafety cabinet? Now we don't have any microscopes or electronic devices in the biosafety cabinet🤨
How can I guarantee that it won't be contaminated？ty very much
Do I need the entire process to be carried out in a biosafety cabinet? Now we don't have any microscopes or electronic devices in the biosafety cabinet
I only want clean buffy coat what should i do
If you are hoping to get the buffy coat layer without residual red blood cells, you can use the EasySep™ RBC Depletion Reagent (Catalog #18170) to deplete RBCs from buffy coats (cdn.stemcell.com/media/files/pis/10000005628-PIS_00.pdf). Feel free to email us at [email protected] if you have any more questions.
Would you recommend aspirating the plasma to obtain a more accurate amount of PBMCs?
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 [email protected].
This is satanic! This must be stopped.
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. Can this protocol be modified for brain regions instead of whole brain
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 [email protected].
This is a great video with a clear and helpful demonstration! Just one thought - to ensure contamination-free growth of your cultures, it's essential to maintain a high level of hygiene inside the laminar flow hood. While the video didn't explicitly cover this, it's a crucial aspect of cell culturing.
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).
They hooked them on the computer now. You can use them for a monthly fee.
The Zika virus is quivering in its boots 🦠
ni
Hi! Is there any reason why the whole blood preparation and the Ficoll shouldn't be mixed before centrifugation? I don't quite undestand this phase because in the centrifugation, they will be mixed anyway. Thanks!
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 [email protected].
What happens if you forget the very last step of washing twice?
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 [email protected].
Let's remove an arm. Are you still a person? Yes, you are. Same for legs, liver, eyes etc. Let's remove a brain. Are you still a person? No. Hence a brain organoid is a person to me. With human rights and everything
May I know the concentration of Matrigel in this protocol?
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 [email protected]
@@STEMCELLTechnologies Thank you.
I wonder... but what if I do know what I want and I want to pursue an academic career but no one would give a chance to pursue a Postdoc position?
Im not a doctor or have any medical background but my question is why you extract the buffy coat with some red blood cells? It should be only the buffy coat.
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 [email protected].
When washing, are the centrifuges at room temperature (20 to 25°C) too? Does the gradient quantity have to be 15ml? If it is 20mL, does it change performance? What is the maximum amount of blood diluted in PBS that can be used per SepMate tube?
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 [email protected].
Hi how can i purchase the kit and magnet please?
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 [email protected].
nice video!
How many rounds can you use this
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 [email protected].
How many different samples can you isolate using one easy sep kit
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 [email protected].
Is the incubator waterless?
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 [email protected].
At 1:21, what is the tool being used to scrape the cell strainer?
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 [email protected].
May i ask? Unfortunately i have no education in microbiology and also English isnt my first. Am i understanding this correct?: Are you using syntetical RNA wich contains code for producing "Yamanaka-factors" wich used to "back-roll" usual cell back to stem cell and put this RNA into empty virus? And what means "self-replicating RNA"? Means it, that this RNA can be found in all copies of next generations of stem cells? If not - how long can this RNA exist inside cell? Is it possible to use for this purpose not artificial RNA, but new artificial chromosome, wich will be able to do the same function and will be able to self-replicate together with cycle of cell division? And also is it possible to control this function (reprogramming to IPS) by light? - firstly add this "thing" to cells and then only later to reprogramm them via turning light on? Sorry about mistakes - my English is pretty bad :( I hope for your answer ;)
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 [email protected].
@@STEMCELLTechnologies Thanks so much for your answer and more thanks for web-links
thank you for the tutorial now i can make my own stem cells and become immortal
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.
Thank you so much❤
Question : I would like to know about the information on the microscope lens used to mark differentiated areas. 2:42 in the video.
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 [email protected].
This is not how it’s described in the protocol, I’m very confused!
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 [email protected].
Yeah you can increase the power of your cells getting used to do difficult things,..
Each time you solve a logical problem you create new brain connections,.. same when you complete a physical challenge for your own achievement,..
A general question regarding contamination: Is it a routine to spray alcohol absolutely on every object that is inserted in the laminar flow cabinet. But how about the flasks that are transported to the microscope for checking stem cell detaching ? I did not see in any video on KZread such an operation of spraying the flask before reintroducing it in the laminar flow cabinet !
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 [email protected].
@@STEMCELLTechnologies thank you .
Question : Regarding the stem cell culture in T 75 flask - using media without CO2 : Can we use a closed cap for T75 flask ?
Hi, we would need more clarification. Please email us at [email protected] on how you're hoping to use this for your application.
What kind of incubator is needed for this process?
You can use a standard CO2 incubator designed for tissue culture work. If you have any more questions, feel free to email us at [email protected].
What is the approximate yield of nucleated cells (absolute number) that you obtain when following this method? Thanks so much.
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 [email protected].
test
what is the difference between this method and the one that used by Shinya Yamanaka? I am kind of confused😅😅
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 [email protected].
@@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
Hi, is macrophage also isolated with this kit?
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 [email protected].
Thank you Dr. Musah!
Thanks for this video!! It Really helped me!!! :D
I need you what'sapp number
Hello, is it possible to use PBS instead of D-PBS?
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 [email protected].
What type of science is this what would I need to study
Hi, this video outlines an enzymatic method to generate a single-cell suspension from mouse spleen prior to the downstream isolation of dendritic cells.
I just learned detailed directions on. How to grow a brain easier than I did doing the brakes on a rav4
Thank you so much for the instructional video. One quick question in the "second wash of isolated PBMCs" section. The video mentions that "if concerned about platelets, run centrifuge at 200 x g for 10 min, with brake-off." 1) Why would reducing the centrifuge speed "protect" platelets? 2) In what circumstances should I be worried about platelets? In what assays/analysis are platelets used for (if there are any examples)?
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 [email protected].
"Promo sm"
Very informative , but the background music is loud and annoying . Plz avoid it or make it minimal ,thanks
Thank you and we'll share this feedback with our team.
Perfect explanation, thanks a lot from Egypt
Great video! Thanks for sharing. Would this technique work for brain tissue of early postnatal mice?
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 [email protected].
@@STEMCELLTechnologies The link is broken. Has it been combined into the NeuroCult Adult CNS kit?
Hi, please try the link again.
What happened after 2018
Please put a video on interferon production
Thank you for your suggestion.