Specimens were collected in various tube types and maintained at room temperature or at 4 °C until testing. Aliquots of fresh whole blood were utilized for real-time antibody labeling and flow cytometric analyses for the Whole Blood (WB) MDSC assay. PBMC were obtained using Lymphoprep™ media and SepMate™ isolation tubes (STEMCELL Technologies, Cambridge, MA).
Sepmate isolation tubes
Manufactured by STEMCELL
Sourced in United States, Canada
SepMate isolation tubes are a specialized laboratory equipment designed for the efficient separation of cells and other biological materials from complex samples. These tubes utilize a specialized buoyant barrier to facilitate the separation process, enabling the isolation of specific cell populations or other target analytes. The SepMate tubes are intended for use in a variety of research and diagnostic applications requiring the separation and isolation of cellular or subcellular components.
Automatically generated - may contain errors
Lab products found in correlation
Lymphoprep media, by STEMCELL (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Rpmi 1640 media, by Corning (1 mentions)
Fetal bovine serum (fbs), by R&D Systems (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Ghost dye red 780, by Cytek Biosciences (1 mentions)
Lsr 2, by BD (1 mentions)
Clone bb23 8e6 8c8, by BD (1 mentions)
Clone 76 2 11, by BD (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Ficoll paque premium density gradient medium, by GE Healthcare (1 mentions)
Ficoll paque density gradient, by STEMCELL (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Cytoflex lx, by Beckman Coulter (1 mentions)
10 protocols using sepmate isolation tubes
1
Blood Specimen Preparation for MDSC
2
Monocyte and PBMC Isolation Protocol
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The human THP-1 monocyte cell line (ATCC TIB-202) was cultured in RPMI-1640 media (Corning, Corning, NY, USA) with 10% heat inactivated FBS (R&D Systems, Minneapolis, MN, USA), an antibiotic-antimycotic (Gibco, Waltham, MA, USA), and 0.05 mM of 2-mercaptoethanol (Gibco). Primary peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers. Briefly, 20 mL of blood was drawn, diluted 1:1 with sterile phosphate buffered saline (PBS) and centrifuged using Lymphoprep density media and SepMate isolation tubes (Stemcell Technologies, Vancouver, BC, USA). The isolated PBMCs were then washed with PBS with 2% inactivated FBS, centrifuged and frozen at −80 °C in RPMI-1640 (Corning) and 10% DMSO. All consent and procedures were approved by the UNCW Institutional Review Board, protocol #21-0168.
3
PBMCs Isolation and Stimulation
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PBMCs were isolated from whole blood of healthy volunteers by density gradient centrifugation using SepMate™ isolation tubes (StemCell Technologies, Cologne, Germany) according to the manufacturer's protocol. Isolated PBMCs were stored at -150°C in medium containing 10% DMSO, 20% FCS, and 70% RPMI until use. For seeding onto polyurethane samples, cells were thawed and resuspended in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) supplemented with 10% off-the-clot serum pooled from male AB blood group donors (H2B, Limoges, France) and seeded at a density of 0.75 × 106 cells in 750 μL medium. Phytohaemagglutinin-L (PHA-L) (15 μg/mL) (Roche, Mannheim, Germany) was used as a positive control to ensure cell functionality. Cultures were incubated at 37°C and 5% CO2 in a humidified atmosphere for 72 h. Following culture with polyurethane samples, 200 μL cell culture medium was collected and centrifuged at 5,000 RPM for 3 min, and the supernatant was stored at -80°C until cytokine analysis.
4
Isolation and Differentiation of Primary Immune Cells
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PBMC were isolated from blood of healthy volunteers using SepMate isolation tubes according to the manufacturer´s instructions (Stemcell Technologies, Vancouver, BC, Canada) after obtaining an appropriate permit from the National Ethical Committee (0120-21/2020/4). Primary mouse BMDMs were isolated from femur and tibia bones of sacrificed mouse for which we obtained the postmortem tissue collection permit from the Administration for Food Safety, Veterinary Sector and Plant Protection (U34401-3/2018/4). Bone marrow was washed out of the bones with a PBS-loaded syringe and was centrifuged for 5 min at 1200 rpm. Erythrocytes were lysed by incubation in NH4Cl buffer for 15 min at 37 °C, followed by centrifugation and aspiration of the supernatant. The pellet was washed in PBS, and after centrifugation was resuspended in DMEM + 20% FBS. We plated 1 × 107 cells per Petri dish and grew them in the presence of MCSF differentiation factor and PenStrep for 6 days. Cells were subsequently frozen.
5
Isolation of PBMC from HGSOC Patients
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Human fresh whole blood from n = 5 HGSOC patients was collected based on the Institutional Review Board of Pontificia Universidad Católica de Chile protocol (ID 190408002, approved 05/07/2020) for studies involving humans. We isolated fresh peripheral blood mononuclear cells (PBMC) through Ficoll gradient in 50 mL SepMate Isolation Tubes (86450, Stemcell Technologies, Vancouver, BC, Canada).
6
Isolation and Staining of Piglet PBLs
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Piglet peripheral blood leukocytes (PBLs) were isolated from cord blood as per manufacturer's protocol with SepMate isolation tubes (Stemcell Technologies, Vancouver, BC). After isolation, PBLs were passed through a 70-μm filter to achieve a single-cell suspension and plated. Antibodies against CD45 (FITC conjugated, Clone K252.1E4; Bio-Rad, Hercules, CA), CD8 (PE conjugated, Clone 76-2-11; BD Biosciences, Franklin Lakes, NJ), CD3 (PE-cy7 conjugated, Clone BB23-8E6-8C8; BD Biosciences), CD4 (PerCP-Cy5.5, Clone 74-12-4; BD Biosciences), and CD335 (APC conjugated, Clone VIV-KM1; Bio-Rad) were used for staining at a dilution of 1:100. Viability was determined with Ghost Dye Red 780 (Tonbo Biosciences, San Diego, CA) at a dilution of 1:1000. BD LSRII and Cytek flow cytometers were used to run the samples. Data were analyzed using FlowJo, v10, software (FlowJo LLC, Ashland, OR).
7
Scalable T-cell Expansion and Transduction
8
Rabbit and Bovine PBMC Isolation
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Rabbit PBMCs were isolated from 5 mL blood collected from the central artery of the rabbit ear before infection and at various time-points post-infection. Immediately after euthanasia, single-cell suspensions were prepared from popliteal lymph nodes and spleen as follows. Tissues were gently disrupted with scissors in sterile RPMI-1640 medium and passed through a 70 μm cell-strainer using a 1-mL syringe plunger. Mononuclear leukocyte suspensions were prepared from peripheral blood and tissue samples using Ficoll-Paque Premium density gradient medium (GE Healthcare). Each 5 mL single-cell suspension was diluted 1:1 in sterile PBS, overlaid onto a 5 mL Ficoll-Paque density cushion, and centrifuged (1825×g) for 20 min at room temperature. Mononuclear leukocytes at the interface were collected and washed in ice-cold PBS before further analysis. Bovine blood was obtained from a healthy donor of CARE-FEPEX ("Cellule d’Appui à la Recherche et à l’Enseignement–Ferme Pédagogique et Experimentale", Dr L. Martinelle) and PBMCs isolated on Ficoll-Paque density gradient using SepMate isolation tubes (STEMCELL technologies).
9
Isolation of Human PBMCs
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Blood from healthy volunteers was collected upon informed consent. PBMCs were obtained by Ficoll gradient centrifugation in Histopaque-1077 (Sigma-Aldrich, St Louis, Missouri, United States) using SepMate™ isolation tubes from STEMCELL™ according to the manufacturer’s instructions. Isolated PBMCs were resuspended in RPMI 1640 supplemented with 10% heat-inactivated FBS.
10
Flow Cytometric Analysis of PBMCs
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Blood samples were taken during clinical routine workup. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll gradient with SepMate isolation tubes (StemCell Technologies) and were cryopreserved in liquid nitrogen. For FC, PBMCs were thawed and resuspended in FC buffer (Phosphate Buffered Saline (PBS)/Bovine Serum Albumin (BSA)/EDTA). Samples were centrifuged for 5 min at 1500 rpm and 4°C and supernatant was discarded. FC buffer was added and cells were transferred to a 96-well plate. Centrifugation was repeated and cells were resuspended in FC buffer containing a FcR Blocking Reagent (Miltenyi Biotec). Cells were incubated for 5 min at room temperature. Next, the following staining antibodies, diluted in FC buffer, were added: anti-CD19 (HIB19), anti-IgD (IA6-2), anti-CD24 (ML5), anti-CD38 (HB-7), anti-CD20 (2H7), anti-CD14 (M5E2), anti-CD3 (SK7), anti-CD56 (HCD56), anti-CD138 (DL-101), anti-CD27 (M-T271), anti-CD21 (Bu32), all from BioLegend. Zombie Aqua Fixable Viability Kit (BioLegend) was used as a viability marker. Incubation was performed for 30 min at 4°C. Afterwards, cells were washed, centrifuged and resuspended in FC buffer. A CytoFLEX LX (Beckman Coulter) was used to acquire data. Analysis was performed with the software ‘Kaluza Flow Cytometry Analysis’ V.2.1 (Beckman Coulter) as illustrated in online supplemental figure 1A .
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