PBS-MINI 바이오리액터 구매에 관심이 있으시면, 한국 유통사인 Bio-Medical Science Co. Ltd.로 이메일을 보내주시기 바랍니다. 이메일 주소는 [email protected]입니다.감사합니다.

Some centrifuges use a digital scale of acceleration and deceleration rather than a basic on/off brake system. In this case, you can set the deceleration to 0. Alternatively, consider using SepMate™ tubes (kzbin.info/www/bejne/Z52WgHqnrdF4kK8 and leaving the brake on, or using the EasySep™ Direct Human PBMC Isolation Kitkzbin.info/www/bejne/joe7mYyjr9tpj80 to skip the centrifuge step altogether. If you have any more questions, feel free to email us at [email protected].

Acceleration rate refers to the increase in speed, while deceleration rate refers to the reduction in speed. In the context of centrifugation, deceleration rate settings are often called braking settings. Some centrifuges offer the capability to modify the acceleration and deceleration settings. For instance, the centrifuge used in this video allows you to choose acceleration settings from 1 (slow acceleration) to 9 (acceleration at maximum power) and deceleration settings from 0 (brake OFF) to 10 (deceleration/brake at maximum power). Using SepMate™ for PBMC isolation by density gradient centrifugation allows you to set the brake ON, reducing the time needed for the isolation of PBMCs compared to using regular tubes. Note that different makes and models of centrifuges may provide different rates of deceleration when braking, and with some models it may be necessary to reduce the deceleration rate to medium or low. If you have any more questions, feel free to email us at [email protected].

After centrifugation, some red blood cells (RBCs) may be present above the SepMate™ insert. These RBCs will not affect performance. RBCs can be more frequent in the PBMC fraction when dealing with older (24hr+) whole blood samples, and we recommend centrifuging the SepMate™ tubes for 20 minutes at 1200 x g (08:00 of the video). After centrifugation, the top layer can be poured off (10:45 of the video), washed once (11:40 of the video), and the cell pellet can be treated with Ammonium Chloride solution. If you have any more questions, feel free to email us at [email protected].

If you does not wish to dilute the blood, you do not need to do so, although recovery might be compromised. This is because when erythrocytes in whole blood are aggregated, some lymphocytes are trapped in the clumps and therefore sediment with the erythrocytes. This tendency to trap lymphocytes is reduced by diluting the blood. Dilution gives a better lymphocyte yield and reduces the size of the cell clumps. Additionally, if you are using SepMate™ tubes, not diluting the blood may result in poor red blood cell depletion and tube overload. If you have any more questions, feel free to email us at [email protected].

The black spots observed in some of the organoids generated with our STEMdiff™ Cerebral Organoid Kit are attributed to retinal pigment epithelium which was reported in the Lancaster et al., 2013 publication (pubmed.ncbi.nlm.nih.gov/23995685/) our kit is based on. This is in alignment with the dorsal anterior characteristics of the organoids. It is common to see pigment in the organoids, however, this varies from cell line to cell line and they arise spontaneously within the organoids. If you have any more questions, feel free to email us at [email protected].

i have read the 05924 protocol which only ask a once wash

Our recommended protocol for density gradient centrifugation (with or without SepMate™ tubes) includes two wash steps. Similarly, our standalone protocol for the RosetteSep™ Human Progenitor Cell Basic Pre-Enrichment Kit (Catalog #15226) lists two washes (1 x 300 x g and 1 x 120 x g). Protocols that require two washes apply to a wider variety of downstream applications that benefit from higher cell purity / reduced platelet contamination. Although the Erythroid Progenitor Reprogramming Kit (Catalog #05924) uses the same RosetteSep™ cocktail that is included in the RosetteSep™ Human Progenitor Cell Basic Pre-Enrichment Kit (Catalog #15226), the Erythroid Progenitor Reprogramming Kit protocol only requires a single 300 x g wash of the enriched hematopoietic progenitors. The 120 x g wash is intentionally excluded from the Erythroid Progenitor Reprogramming Kit protocol because it has an intermediate expansion step that increases the number of erythroid progenitors before starting the reprogramming step. The additional wash at 120 x g that is normally included to reduce platelet contamination would therefore not improve the performance of the downstream workflow in this particular application. Additionally, washes inherently result in some cell loss, so excluding the wash step helps retain more starting cells that can then be used for downstream reprogramming. If you have any more questions, feel free to email us at [email protected].

How can I guarantee that it won't be contaminated？ty very much

We recommend using a microscope inside a Level 2 biological safety cabinet when isolating putative iPSC colonies during reprogramming. The biosafety cabinet helps to maintain the sterility of the sample and protects users from the cells (which should be treated as potentially infectious). The microscope is recommended because the magnification improves accuracy during colony picking, reducing the transfer of non-reprogrammed cells (fibroblasts, partially reprogrammed cells, or differentiated cells) that could otherwise negatively impact your initial iPSC culture quality. If you have any more questions, feel free to email us at [email protected].

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.

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].

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 [email protected].

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 [email protected].

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].

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.

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].

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, 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].

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].

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].

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].

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].

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

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 [email protected].

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].

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 [email protected].

@@STEMCELLTechnologies thank you .

Hi, we would need more clarification. Please email us at [email protected] 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 [email protected].

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].

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

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].

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].