Sepmatetm tube

Manufactured by STEMCELL

Sourced in Canada, Germany

SepMateTM tubes are a specialized laboratory equipment designed for the separation of cells, tissues, or other biological samples. They utilize a proprietary density gradient medium to facilitate efficient and reproducible cell separation. The core function of SepMateTM tubes is to enable the isolation and purification of target cell populations from complex mixtures, supporting various downstream applications in life science research and clinical diagnostics.

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Lab products found in correlation

Lymphoprep, by STEMCELL (2 mentions) Lymphopreptm, by STEMCELL (2 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions) Lunatm cell counting slides, by Logos Biosystems (1 mentions) Lunatm automated cell counter, by Logos Biosystems (1 mentions) Ficoll plaque premium, by GE Healthcare (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Sfem medium, by STEMCELL (1 mentions) Histopaque 1077 hybri maxtm, by Merck Group (1 mentions) Easyseptm human cd8 t cell enrichment kit, by STEMCELL (1 mentions) Trypan blue, by Merck Group (1 mentions) Fetal calf serum (fcs), by Merck Group (1 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Celltracker green cmfda dye, by Thermo Fisher Scientific (1 mentions) Cytation 1 cell imaging multi mode reader, by Agilent Technologies (1 mentions) Cd14 microbead, by Miltenyi Biotec (1 mentions) Tc20 counter, by Bio-Rad (1 mentions) Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Ficoll paquetm premium, by GE Healthcare (1 mentions)

13 protocols using sepmatetm tube

Isolation of PBMCs from Sepsis Patients

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Human PBMCs were isolated from the blood of sepsis patients and healthy donors. The use of PBMCs complied with institutional ethics guidelines and approved protocols of Jinling Hospital. PBMCs were collected from patients with sepsis and HCs by density gradient centrifugation. Briefly, 4 mL of Lymphoprep^TM (07801, STEMCELL) was added to a 15-mL SepMate^TM tube (85415, STEMCELL). Two milliliters of blood were diluted with 2 mL of PBS, and the blood was layered on top of Lymphoprep^TM. Next, the mixture was centrifuged at 800 × g for 20 min at room temperature with the brake off. The upper plasma and PBMC layers were removed by centrifugation at 800 × g for 3 min at room temperature for subsequent analyses.

Wu J., Liu Q., Zhang X., Tan M., Li X., Liu P., Wu L., Jiao F., Lin Z., Wu X., Wang X., Zhao Y, & Ren J. (2022). The interaction between STING and NCOA4 exacerbates lethal sepsis by orchestrating ferroptosis and inflammatory responses in macrophages. Cell Death & Disease, 13(7), 653.

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PBMC Isolation from Whole Blood

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In brief, 3 ml of Lymphoprep (07801, STEMCELL technologies, Vancouver, BC, Canada) was added to a 15-ml SepMate^TM tube (85415, STEMCELL technologies, Vancouver, BC, Canada). Two milliliters of blood was diluted by mixing with 2 ml of phosphate-buffered saline (PBS), and the blood was overlaid on the Lymphoprep^TM. Next, the mixture was centrifuged at 800 × g for 20 min at room temperature (RT) without braking. The upper plasma layer was removed, and the PBMC layer in the plasma was retained.

Liu Q., Wu J., Zhang X., Li X., Wu X., Zhao Y, & Ren J. (2021). Circulating mitochondrial DNA-triggered autophagy dysfunction via STING underlies sepsis-related acute lung injury. Cell Death & Disease, 12(7), 673.

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Isolation of PBMCs from Human Samples

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The collection of human blood samples and intestinal tissue was from our previous study.¹³ Sepsis and septic shock were defined according to previous reports.¹⁹ PBMC isolation was performed using Lymphoprep (07801; STEMCELL) and SepMateTM tube (85415; STEMCELL) according to the manufacturer's instructions.

Zhang X., Wu J., Liu Q., Li X., Yang Y., Wu L., Wu X., Zhao Y, & Ren J. (2023). RIPK3–MLKL necroptotic signalling amplifies STING pathway and exacerbates lethal sepsis. Clinical and Translational Medicine, 13(7), e1334.

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Isolation and Enumeration of PBMCs

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The collected whole blood (1.5 mL) samples were diluted in 2.5 mL of Hank’s balanced salt solution (HBSS) and carefully layered into 15 mL SepMate^TM tubes (StemCell Technologies, Vancouver, BC, Canada) containing 3.5 mL Ficoll Plaque Premium (GE Healthcare, Chicago, IL, USA). The tubes were centrifuged for 15 min (min) at 1200× g, 20 °C. The upper layer consisting of PBMCs was decanted into a 15 mL falcon tube containing 7 mL of HBSS. Following mixing, the tubes were centrifuged at 400× g (18 °C) for 15 min. Following centrifugation, the supernatant was discarded, and the pellet was resuspended in 7 mL of HBSS. The resuspended cells were centrifuged again under the same conditions. Then, the supernatant was discarded, and the pellet was resuspended in Roswell Park Memorial Institute medium (RPMI; Gibco, Carlsbad, CA, USA) containing 1% l-glutamine, 1% of antibiotic (100 U/mL penicillin and 100 µg/mL streptomycin) and 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). The PBMCs in each sample were counted using LUNA^TM Cell Counting Slides (Logos Biosystems, Annandale, VA, USA) with the Luna^TM Automated Cell Counter (Logos Biosystems, Annandale, VA, USA).

Barboza-Solis C., Najimudeen S.M., Perez-Contreras A., Ali A., Joseph T., King R., Ravi M., Peters D., Fonseca K., Gagnon C.A., van der Meer F, & Abdul-Careem M.F. (2021). Evaluation of Recombinant Herpesvirus of Turkey Laryngotracheitis (rHVT-LT) Vaccine against Genotype VI Canadian Wild-Type Infectious Laryngotracheitis Virus (ILTV) Infection. Vaccines, 9(12), 1425.

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Generation of iPSCs from RTT Patients

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Peripheral blood mononuclear cells (PBMCs) from RTT patients recruited for the study were collected and isolated with SEPMATE ^TM tubes (STEMCELL) according to the protocol. Erythroblast population was enriched by maintaining PBMCs for 10 days in SFEM Medium (STEMCELL) and then transducing with Cytotune 2.0 Sendai Reprogramming Kit (Life Technologies). After about 3 weeks the first iPSC colonies appeared on the mouse embryonic fibroblast (MEF) layer. Single clones were expanded and characterized for stemness and genomic stability.
Pluripotency marker expression was confirmed through immunofluorescence (using antibody against OCT3/4 and TRA-1-60) and RT-PCR (with primers for OCT3/4, SOX2 and NANOG). Karyotype analysis on at least 20 metaphases/sample allowed visualization of chromosome aberrations occurring during reprogramming. Refer to [38 (link)] for further details.

Perego S., Alari V., Pietra G., Lamperti A., Vimercati A., Camporeale N., Garzo M., Cogliati F., Milani D., Vignoli A., Peron A., Larizza L., Pizzorusso T, & Russo S. (2022). Modeling RTT Syndrome by iPSC-Derived Neurons from Male and Female Patients with Heterogeneously Severe Hot-Spot MECP2 Variants. International Journal of Molecular Sciences, 23(22), 14491.

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Peripheral Blood Mononuclear Cell Isolation

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Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using Histopaque®-1077 Hybri-Max^TM (Sigma-Aldrich) density gradient centrifugation in SepMate^TM tubes (Stemcell) as previously described (20, 23, 24). Isolated PBMCs were cryopreserved in fetal calf serum containing 10% DMSO and stored in liquid nitrogen.

Reynolds C.J., Pade C., Gibbons J.M., Otter A.D., Lin K.M., Muñoz Sandoval D., Pieper F.P., Butler D.K., Liu S., Joy G., Forooghi N., Treibel T.A., Manisty C., Moon J.C., Semper A., Brooks T., McKnight Á., Altmann D.M., Boyton R.J., Abbass H., Abiodun A., Alfarih M., Alldis Z., Altmann D.M., Amin O.E., Andiapen M., Artico J., Augusto J.B., Baca G.L., Bailey S.N., Bhuva A.N., Boulter A., Bowles R., Boyton R.J., Bracken O.V., O’Brien B., Brooks T., Bullock N., Butler D.K., Captur G., Carr O., Champion N., Chan C., Chandran A., Coleman T., Couto de Sousa J., Couto-Parada X., Cross E., Cutino-Moguel T., D’Arcangelo S., Davies R.H., Douglas B., Di Genova C., Dieobi-Anene K., Diniz M.O., Ellis A., Feehan K., Finlay M., Fontana M., Forooghi N., Francis S., Gibbons J.M., Gillespie D., Gilroy D., Hamblin M., Harker G., Hemingway G., Hewson J., Heywood W., Hickling L.M., Hicks B., Hingorani A.D., Howes L., Itua I., Jardim V., Lee W.Y., Jensen M., Jones J., Jones M., Joy G., Kapil V., Kelly C., Kurdi H., Lambourne J., Lin K.M., Liu S., Lloyd A., Louth S., Maini M.K., Mandadapu V., Manisty C., McKnight Á., Menacho K., Mfuko C., Mills K., Millward S., Mitchelmore O., Moon C., Moon J., Muñoz Sandoval D., Murray S.M., Noursadeghi M., Otter A., Pade C., Palma S., Parker R., Patel K., Pawarova M., Petersen S.E., Piniera B., Pieper F.P., Rannigan L., Rapala A., Reynolds C.J., Richards A., Robathan M., Rosenheim J., Rowe C., Royds M., Sackville West J., Sambile G., Schmidt N.M., Selman H., Semper A., Seraphim A., Simion M., Smit A., Sugimoto M., Swadling L., Taylor S., Temperton N., Thomas S., Thornton G.D., Treibel T.A., Tucker A., Varghese A., Veerapen J., Vijayakumar M., Warner T., Welch S., White H., Wodehouse T., Wynne L., Zahedi D., Chain B, & Moon J.C. (2022). Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure. Science (New York, N.y.).

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Isolation of PBMCs from Young Sleeve Gastrectomy Patients

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Whole blood was collected from young sleeve gastrectomy patients (15–21 years old). The Sepmate^TM tubes (Stemcell) and Lymphoprep^TM (Stemcell) were used to separate out red blood cells and platelets according to manufacturer’s protocol. Briefly, 50 mL Sepmate^TM tubes were filled at the bottom with 15 mL of Lymphoprep^TM. Whole blood was diluted with phosphate-buffered saline containing 2% fetal bovine serum. Diluted whole blood was added to the tube containing Lymphoprep^TM. The tubes were centrifuged at 1200 g for 10 minutes. Following this supernatant was poured into a separate tube. The supernatant was diluted with phosphate-buffered saline containing 2% fetal bovine serum. The supernatant was centrifuged at 300 g for 8 minutes. The supernatant was discarded, and the pellet was re-suspended in phosphate-buffered saline containing 2% fetal bovine serum. The pellet was centrifuged at 120 g for 10 minutes. The resulting peripheral blood mononuclear cells were cultured in dendritic cell media or put through the negative selection EasySep^TM Human CD8+ T cell Enrichment Kit (Stemcell).

Holokai L., Chakrabarti J., Broda T., Chang J., Hawkins J.A., Sundaram N., Wroblewski L.E., Peek RM J.r., Wang J., Helmrath M., Wells J.M, & Zavros Y. (2019). Increased Programmed Death-Ligand 1 is an Early Epithelial Cell Response to Helicobacter pylori Infection. PLoS Pathogens, 15(1), e1007468.

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Isolation and Cryopreservation of PBMCs

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PBMCs were isolated from 50 mL of venous blood by density gradient centrifugation using Ficoll-paque plus (GE Healthcare) and SepMateTM tubes (STEMCELL) within 24 h of blood collection. PBMCs were washed twice with phosphate buffered saline (PBS) and counted using Türks solution and checked for viability with Trypan Blue (both Sigma Aldrich). PBMCs were frozen in medium containing RPMI (Gibco), 20% FCS (LPS)‚ and 10% DMSO (Sigma Aldrich) and stored in liquid nitrogen until further use.

Imhof C., Messchendorp A.L., van der Heiden M., Baan C.C., van der Molen R.G., Remmerswaal E.B., de Vries R.D., Diavatopoulos D.A., Boerma A., Bakker F.J., Oosterhout E., Bemelman F.J., Hilbrands L.B., Reinders M.E., Gansevoort R.T., Sanders J.S, & van Baarle D. (2022). SARS-CoV-2 Spike-specific IFN-γ T-cell Response After COVID-19 Vaccination in Patients With Chronic Kidney Disease, on Dialysis, or Living With a Kidney Transplant. Transplantation Direct, 8(11), e1387.

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Isolation and Cryopreservation of PBMCs

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At 9 weeks of life and at necropsy blood was collected in heparinized tubes and PBMCs was enriched the same day using routine protocols (layering blood over high density Ficoll gradient and centrifugation at 1200g for 10 min in SepMate^TM tubes (STEMCELL Technologies Germany GmbH, Köln, Germany). Leukocytes were washed extensively in Phosphate buffered saline (PBS) (no calcium, no magnesium). The pellet was resuspended in cold freezing medium (containing 50% RPMI1640 Medium, 40% FCS and 10% DMSO), dispensed in 2ml cryovials, and transferred to -70°C freezer and after 24h transferred into -150°C freezer.

Kraft C., Hennies R., Dreckmann K., Noguera M., Rathkjen P.H., Gassel M, & Gereke M. (2019). Evaluation of PRRSv specific, maternally derived and induced immune response in Ingelvac PRRSFLEX EU vaccinated piglets in the presence of maternally transferred immunity. PLoS ONE, 14(10), e0223060.

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Monocyte Adhesion Assay with PBMCs

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Deidentified human peripheral blood mononuclear cells (PBMCs) were obtained through leukocyte reduction system chambers/cones routinely collected at City of Hope after plateletpheresis. The research consents were obtained from the donors’ next of kin and ethical approval for this study was granted by the Institutional Review Board of City of Hope (IRB #09025). PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (GF Healthcare) using SepMate^TM tubes (STEMCELL Technologies Inc.) according to the protocol provided by the manufacturer. Subsequently, monocytes were isolated from PBMCs by immunomagnetic positive selection of CD14⁺ cells using CD14 microbeads (Miltenyi Biotec). Monocytes adhesion assay was performed using the isolated monocytes from four different healthy donors, which were labeled with CellTracker™ Green CMFDA Dye (Thermo Fisher Scientific) and incubated with monolayer ECs (4 × 10³ cells per cm²) for 15–30 min in a cell culture incubator. The nonattached monocytes were then washed off with complete EC growth medium. The attached monocyte numbers were evaluated on Cytation™ 1 Cell Imaging Multi-Mode Reader (BioTek) using green fluorescent channel. Average numbers per sample were calculated from five randomly selected fields.

Calandrelli R., Xu L., Luo Y., Wu W., Fan X., Nguyen T., Chen C.J., Sriram K., Tang X., Burns A.B., Natarajan R., Chen Z.B, & Zhong S. (2020). Stress-induced RNA–chromatin interactions promote endothelial dysfunction. Nature Communications, 11, 5211.

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