Sepmate 50 tube

Manufactured by STEMCELL

Sourced in Canada, United Kingdom, Germany

SepMate-50 tubes are laboratory equipment designed to facilitate the separation and isolation of cells from biological samples. They feature a specialized porous barrier that enables efficient separation of cells during centrifugation, promoting the isolation of targeted cell populations.

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Lab products found in correlation

Ficoll paque plus, by GE Healthcare (14 mentions) Lymphoprep, by STEMCELL (13 mentions) Histopaque 1077, by Merck Group (5 mentions) Rneasy mini kit, by Qiagen (4 mentions) Lymphoprep density gradient medium, by STEMCELL (4 mentions) Ficoll paque premium, by GE Healthcare (4 mentions) Lymphoprep solution, by STEMCELL (3 mentions) Fetal bovine serum (fbs), by Omega Scientific (3 mentions) Human lympholyte separation medium, by Cedarlane (3 mentions) Vacutainer, by BD (3 mentions) Ack lysis buffer, by Thermo Fisher Scientific (2 mentions) 2100 bioanalyzer, by Agilent Technologies (2 mentions) Rnase free dnase set, by Qiagen (2 mentions) Rna 6000 pico kit, by Agilent Technologies (2 mentions) Ficoll pacque gradient, by Thermo Fisher Scientific (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Fetal calf serum (fcs), by Merck Group (2 mentions) Biocoll, by Merck Group (2 mentions) Gm csf, by Thermo Fisher Scientific (2 mentions) Ls column, by Miltenyi Biotec (2 mentions)

Close spelling variants of this product

SepMate tubes (281 citations) SepMate (68 citations) SepMate-50 (41 citations) SepMate 50 mL tubes (7 citations) SepMateTM-50 separation tubes (6 citations) SepMate 50-mL conical tubes (3 citations) SepMate-50 PBMC Isolation tubes (3 citations) SepMate-50 (IVD) tubes (2 citations)

86 protocols using sepmate 50 tube

Isolation and Analysis of PBMCs from COVID-19 Patients

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Blood samples were collected up to 5 times over 4 weeks. Heparin-containing tubes were used to collect peripheral blood samples from patients with COVID-19. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Little Chalfont, UK) and SepMate-50 tubes (STEMCELL Technologies, Vancouver, Canada). PBMC isolation was performed according to the manufacturer's instructions of SepMate-50 tubes (STEMCELL Technologies). PBMC samples were stored with CELLBANKER (ZENOGEN PHARMA, Fukushima, Japan) in a freezer at -80 °C. TRIzol LS (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract total RNA and SARS-CoV-2 inactivation. Total RNA was extracted and purified with an RNeasy Mini Kit (Qiagen, Hiden, Germany) according to the manufacturer's instructions. The RNA amounts and purity were measured with an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA).

Funakoshi Y., Ohji G., Yakushijin K., Ebisawa K., Arakawa Y., Saegusa J., Matsumoto H., Imanishi T., Fukuda E., Matsutani T., Mori Y., Iwata K, & Minami H. (2021). Massive surge of mRNA expression of clonal B-cell receptor in patients with COVID-19. Heliyon, 7(8), e07748.

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Isolation of PBMCs from Whole Blood

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Following venipuncture, PBMCs were isolated using SepMate -50 tubes (StemCell Technologies), as per the manufacturer's protocol. The blood from each subject was poured into 50 ml tubes (Falcon, ThermoFisher) and carefully diluted 1:1 with a Dulbecco's PBS (D-PBS, Gibco by Life Technologies) containing 2% fetal bovine serum solution. Then, 15 ml Lymphoprep (StemCell Technologies) was transferred into a SepMate -50 tube (StemCell Technologies) and the diluted sample subsequently added to the Lymphoprep-filled tube, by carefully pipetting down the side of the tube. After centrifugation (1 200 x g, 10 minutes, RT), the plasma layer was removed and the PBMCs poured off into a sterile 50 ml tube (Falcon, ThermoFisher) containing D-PBS (Gibco by Life Technologies). The tube was subsequently filled with cold D-PBS until a total volume of 50 ml and centrifuged (300 x g, 10 minutes, 4°C).
The supernatant was removed, and the pellet gently resuspended in D-PBS (Gibco by Life Technologies), using a transfer pipette (Sarstedt). The sample was then filled up to 50 ml with cold D-PBS, centrifuged (220 x g, 5 minutes, 4°C), and the supernatant discarded. In parallel, IGRA status was determined on whole-blood samples using QuantiFERON-TB Gold (Cellestis) following the manufacturer's instructions.

Das J., Idh N., Pehrson I., Paues J., & Lerm M. (2021). A DNA methylome biosignature in alveolar macrophages from TB-exposed individuals predicts exposure to mycobacteria.

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Macrophage Differentiation and Stimulation

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Buffy coats from healthy donors were obtained from Karolinska University Hospital (Solna, Stockholm), the peripheral blood mononuclear cells (PBMCs) were isolated via gradient centrifugation using Ficoll-Paque Plus (17144002, Cytiva) in SepMate^TM-50 tubes (85450, Stemcell Technologies). The CD14⁺CD16^- monocytes were isolated using the Human Classical Monocyte Isolation Kit (130-117-337, Miltenyi Biotec) by following the kit instructions. The magnetically labeled non-target cells were depleted using a MACS® column while the unlabeled CD14⁺CD16^- monocytes ran through the column and were collected. To differentiate human macrophages from the isolated monocytes, 0.5 × 10⁶ monocytes/well were seeded in 24-well plate with the culture medium RPMI 1640 GlutaMAX™ medium (Gibco) containing 10% FBS, 1% penicillin-streptomycin and 40 ng/ml of recombinant human M-CSF (PHC9504, Thermo Fisher Scientific). Cells were cultured for 10 days at 37 °C in conventional CO₂ incubator with changes of fresh culture medium every 3–4 days. On day 10, differentiated macrophages were stimulated by LPS (250 ng/ml) or PBS together with indicated hIgG1 E4 or M2139 immune complexes (10 µg/ml) overnight. The supernatant was then collected for cytokine detection.

He Y., Ge C., Moreno-Giró À., Xu B., Beusch C.M., Sandor K., Su J., Cheng L., Lönnblom E., Lundqvist C., Slot L.M., Tong D., Urbonaviciute V., Liang B., Li T., Lahore G.F., Aoun M., Malmström V., Rispens T., Ernfors P., Svensson C.I., Scherer H.U., Toes R.E., Gjertsson I., Ekwall O., Zubarev R.A, & Holmdahl R. (2023). A subset of antibodies targeting citrullinated proteins confers protection from rheumatoid arthritis. Nature Communications, 14, 691.

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Isolating PBMCs from Blood Samples

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Blood was harvested from matched patients undergoing an ASCC biopsy, with between 15 and 30 ml collected in EDTA tubes for processing to obtain PBMCs. Density gradient SepMateTM-50 tubes (Stemcell Technologies, Vancouver, BC, Canada) containing Ficoll-Paque PLUS (GE Healthcare Life Sciences, Mississauga, ON, Canada) were used with blood diluted 1:2 with phosphate-buffered saline (PBS), centrifuged at 1200 × g for 10 min and the mononuclear cells in the top layer collected, washed (PBS) and re-centrifuged. An ammonium-chloride-potassium (ACK) lysis was undertaken with 3 ml of ACK lysis buffer (Gibco, Thermo Fisher Scientific) for 3 min to remove residual red blood cells before the cells were washed, centrifuged and cryopreserved for downstream genomic analysis.

Guerra G.R., Kong J.C., Millen R.M., Read M., Liu D.S., Roth S., Sampurno S., Sia J., Bernardi M.P., Chittleborough T.J., Behrenbruch C.C., Teh J., Xu H., Haynes N.M., Yu J., Lupat R., Hawkes D., Di Costanzo N., Tothill R.W., Mitchell C., Ngan S.Y., Heriot A.G., Ramsay R.G, & Phillips W.A. (2021). Molecular and genomic characterisation of a panel of human anal cancer cell lines. Cell Death & Disease, 12(11), 959.

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Isolation and Characterization of PBMCs

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Peripheral blood mononuclear cells (PBMC) of 50 healthy blood donors were isolated using Ficoll-Paque^TM Plus (GE Healthcare), Dulbecco’s phosphate buffered saline (DPBS) CTSTM (Gibco Life Technologies), fetal bovine serum (FBS, Sigma), and SepMate^TM-50 tubes according to the manufacturer’s protocol (Stemcell Technologies). Isolated cell counts were determined using NucleoCounter^® NC-100^TM (chemometec). The use of anonymized PBMCs from blood donors is in accordance with the rules of the Finnish Supervisory Authority for Welfare and Health (Valvira). All RNA was extracted from 1 to 10 × 10⁶ cells using RNeasy Mini kit and Rnase-Free DNAse Set (both Qiagen), and the quantity was measured using The Qubit^TM RNA High Sensitivity Assay Kit in Qubit^® 2.0 fluorometer (ThermoScientific). The quality of RNA was assessed with an RNA 6000 Pico Kit (Agilent Genomics) in a 2100 Bioanalyzer (Agilent Genomics). Samples with RNA integrity number (RIN) value over 8.0 were used in the library preparation.

Johansson T., Koskela S., Yohannes D.A., Partanen J, & Saavalainen P. (2021). Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes. Frontiers in Genetics, 12, 635601.

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Isolation of Peripheral Blood Mononuclear Cells

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The bottom of SepMate-50 tubes (STEMCELL 85450) were filled with 4.5 ml of Ficoll-Paque PLUS (GE Healthcare 17–1440-03). EDTA treated whole blood was diluted at a 1:1 ratio with 2% FBS (Gibco 11875–093) in RPMI media (Hyclone SH30070.03) before slowly pipetted into the previously prepared SepMate tubes. Tubes were then centrifuged at 1,200g with no breaks for 10 min. The supernatant was collected into a new 15 ml tube and spun for 300g for 8 min with breaks. The supernatant was removed and the pelleted cells were washed with 2% FBS in RPMI before spun down once again. The supernatant was removed and the pellet was suspended in 1 ml of PBS (Sigma RNBK1816).

Schartl M., Schlupp I., Schartl A., Meyer M.K., Nanda I., Schmid M., Epplen J.T, & Parzefall J. (1991). On the stability of dispensable constituents of the eukaryotic genome: stability of coding sequences versus truly hypervariable sequences in a clonal vertebrate, the amazon molly, Poecilia formosa.. Proceedings of the National Academy of Sciences of the United States of America, 88(19), 8759-8763.

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Isolation and Cryopreservation of Mononuclear Cells

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Blood samples were collected from healthy adult donors with informed consent in accordance with guidelines from the Duke Institutional Review Board committee. Mononuclear cells were isolated by Ficoll-paque plus (GE) density gradient centrifugation with SepMate-50 tubes (STEMCELL Technologies). Cells were cryopreserved in liquid nitrogen until use. For microbial antigen-specific T-cell studies, blood samples were collected 2 to 5 weeks after tetanus-diphtheria boost and/or influenza vaccination.

Su K.Y., Watanabe A., Yeh C.H., Kelsoe G, & Kuraoka M. (2016). Efficient Culture of Human Naïve and Memory B cells for Use as Antigen-presenting Cells. Journal of immunology (Baltimore, Md. : 1950), 197(10), 4163-4176.

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PBMC Isolation from Seropositive and Seronegative Subjects

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Peripheral blood from 20 seronegative and 20 seropositive subjects (based on anti-Spike IgG ELISA) was collected in EDTA-coated tubes and stored at 4 °C prior to processing. Blood was diluted 1:1 with PBS containing 2% FCS and 1 mM EDTA. Peripheral blood mononuclear cells (PBMCs) were isolated using a density gradient method. Lymphoprep solution (Stem Cell) was pipetted to the bottom of SepMate-50 tubes (Stem Cell), and diluted blood carefully layered on top. After 10 min centrifugation at 1200 × g, the upper phase containing mononuclear cells was transferred to a new tube. PBMCs were washed twice with PBS with 2% FCS and 1 mM EDTA. After counting, cells were aliquoted, frozen in FCS containing 10% DMSO, and stored at −80 °C.

Stanczak M.A., Sanin D.E., Apostolova P., Nerz G., Lampaki D., Hofmann M., Steinmann D., Krohn-Grimberghe M., Thimme R., Mittler G., Waller C.F., Pearce E.J, & Pearce E.L. (2021). IL-33 expression in response to SARS-CoV-2 correlates with seropositivity in COVID-19 convalescent individuals. Nature Communications, 12, 2133.

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Isolation of Monocytes from Human Whole Blood

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Human whole blood (WB) was obtained from healthy volunteers as part of a lung cancer metabolism study according to a University of Kentucky IRB approved protocol (IRB# 43807). Monocytes were isolated from WB using the RosetteSep™ human monocyte enrichment cocktail kit (StemCell Technologies Inc., Cambridge). Each mL of WB was mixed and incubated for 20 min with 2 μL of 0.5 mM EDTA, and 50 μL of the RosetteSep™ monocyte negative selection cocktail. The WB was diluted 1:1 with phosphate-buffered saline (PBS) (Ca²⁺ and Mg²⁺ free) + 1× antibiotic-antimycotic (Thermo fisher) + 2 % endotoxin-free fetal bovine serum (FBS) and then added to a Ficoll-Hypaque (GE) gradient in SepMate™-50 tubes (StemCell Technologies Inc.), followed by centrifugation at 1,200 ×g for 20 min. The top layers were pooled and then spun for 10 min at 400 ×g. The resulting cell pellets were resuspended in the same 2 % FBS-PBS dilution buffer and washed twice via centrifugation at 600 ×g and 120 ×g for 10 minutes each. Finally, the cells were counted and seeded into 6-well plates at a density of 3 – 5 × 10⁶ cells per well in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 0.2 % glucose, 2 mM glutamine, 10 % FBS, 1× antibiotic-antimycotic, and 50 ng/mL of monocyte colony stimulating factor (M-CSF) (M0 medium).

Kang W.Y., Thompson P.T., El-Amouri S.S., Fan T.W., Lane A.N, & Higashi R.M. (2019). Improved Segmented-Scan Spectral Stitching for Stable Isotope Resolved Metabolomics by Ultra-High-Resolution Fourier Transform Mass Spectrometry. Analytica chimica acta, 1080, 104-115.

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Trap-15 Reinvigorates TGFβ1-Suppressed PBMC

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Example 8

A cell based assay is performed to determine whether Trap-15 could reinvigorate human PBMC function in the presence of immune suppressor TGFβ 1.

Human PBMC was obtained from healthy donors. PBMC were isolated in SepMate-50 tubes (StemCell Technologies) containing Lymphoprep density gradient reagent (StemCell Technologies). 96-well plates (Corning, Cat #3799) were coated with 0.5 μg/mL functional-grade anti-CD3 (eBioscience, Cat #16-0037-85) in PBS at 4° C. overnight. On the next day, the coated plates were washed twice with DPBS buffer (Hyclone, Cat #SH30256.01). 1×10⁵/well PBMC were added to the plates. Serial diluted Trap-15, M7824, anti-PD-1 antibody (antibody 21F12-1F6) and IgG control negative control IgG (heavy chain: SEQ ID NO:22; light chain: SEQ ID NO:23) were mixed with human TGFβ1 (SinoBiological, Cat #10804-HNAC) separately and added to the plate, the final concentration of TGFβ1 in plate is 5 ng/mL. After cell culture at 37° C. for 72 h, supernatants were collected and tested for IFNγ levels with ELISA kit (R&D Systems, Cat #DY285B). Results were shown in FIG. 6.

The results indicated that Trap-15 could block both PD-1 and TGFβ1, reinvigorated the PBMCs, elevated the IFNγ levels in a dose dependent manner. Surprisingly, M7824 did not show any effect in this assay system.

US11802145B2. Recombinant protein targeting PD-1 and TGFß (2023-10-31). Nanjing Leads Biolabs Co., Ltd. [CN]. Inventors: Xiaoqiang Kang [US], Xiao Huang [CN].

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