Healthy adult blood was collected from residual leukocyte packs following blood donation (ARUP, Sandy, UT), and PBMC's were isolated by density gradient centrifugation (Lymphoprep, StemCell Technologies). Residual palatine tonsil samples were acquired from pediatric patients, ages 2-16, undergoing routine tonsillectomies for tonsillar hyperplasia or recurrent tonsillitis. All tonsil samples were stored on ice in complete media (RPMI 1640 with 10% FBS, 1% penicillin/streptomycin, and 15 mM HEPES) (R10) and processed within 2 hours of surgery. 1 cm x 1 cm pieces were cut and frozen in O.C.T. Compound (Fisher) at -80°C for later use. The remaining tonsil tissue was minced in media and dissociated using a 100 µM cell strainer. Tonsil mononuclear cells were isolated by density gradient centrifugation (Lymphoprep, StemCell Technologies). PBMC and tonsil mononuclear cells were frozen in media containing 25% FBS and 10% DMSO at -80°C for later use. All human samples were deidentified prior to receipt, and the research protocol was deemed exempt by the University of Utah Institutional Review Board (Protocol 100683).
Lymphoprep
Manufactured by STEMCELL
Sourced in Canada, United Kingdom, United States, France, Germany, Norway, China, Australia, Singapore, Italy
Lymphoprep is a density gradient medium used for the isolation of lymphocytes from whole blood or buffy coat. It separates lymphocytes from other blood cells based on their density.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (51 mentions)
Sepmate tube, by STEMCELL (46 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (41 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (36 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (24 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (23 mentions)
Rpmi 1640, by Thermo Fisher Scientific (21 mentions)
Penicillin, by Thermo Fisher Scientific (20 mentions)
Sepmate, by STEMCELL (20 mentions)
L glutamine, by Thermo Fisher Scientific (19 mentions)
Glutamax, by Thermo Fisher Scientific (18 mentions)
Streptomycin, by Thermo Fisher Scientific (16 mentions)
L glutamine, by Merck Group (15 mentions)
Penicillin, by Merck Group (15 mentions)
Rpmi 1640, by Merck Group (14 mentions)
Human ab serum, by Merck Group (14 mentions)
Streptomycin, by Merck Group (14 mentions)
Leucosep tube, by Greiner (14 mentions)
Sepmate 50 tube, by STEMCELL (13 mentions)
Sepmate 50, by STEMCELL (12 mentions)
977 protocols using lymphoprep
1
Isolation and Cryopreservation of PBMC and Tonsil Cells
2
PBMC and Plasma Isolation from Murine Blood
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At the defined time points, retro-orbital blood was collected from mice. The isolation of PBMCs and plasma was achieved via centrifugation using SepMate-15 and Lymphoprep gradient medium (StemCell Technologies). 200 μl blood was immediately diluted with 800 μl PBS with 2% FBS. The blood diluent was then added to SepMate-15 tubes with 6ml Lymphoprep (StemCell Technologies). Centrifugation at 1200 g for 20 minutes was used to isolate RBCs, PBMCs and plasma. 250ul diluted plasma was collected from the surface layer. The remaining solution at the top layer was poured to a new tube to isolate PBMCs, which were washed once with PBS + 2% FBS. The separated plasma was used in ELISA and neutralization assay.
3
Isolation of Bovine PBMC for RNA Analysis
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Blood samples were collected from the jugular vein of the dairy cows into 10 mL K2-EDTA vacutainer tubes (BD Vacutainer, Becton Dickinson Co., Franklin Lakes, NJ, USA). Two tubes were collected from each animal to obtain 15 to 20 mL of blood, and the samples were immediately placed on ice and transported to the laboratory for the isolation of PBMC within 30 min of the time of sampling.
The PBMCs were isolated using density gradient centrifugation. Briefly, whole blood samples were diluted with phosphate-buffered saline (PBS) in a 1:1 ratio in 15 mL conical tubes. Then, 4 mL of Lymphoprep (STEMCELL Technologies lnc., Vancouver, BC, Canada) was added to the new tubes, and 8 mL of the diluted blood samples were overlaid on the Lymphoprep. After centrifugation for 20 min at 800× g and at 22 °C, the layer of cells above the Lymphoprep was collected and washed twice with PBS to obtain purified PBMCs that were then suspended in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and transferred to a 1.5 mL tube. The PBMCs were immediately stored at −80 °C until the RNA isolation process.
The PBMCs were isolated using density gradient centrifugation. Briefly, whole blood samples were diluted with phosphate-buffered saline (PBS) in a 1:1 ratio in 15 mL conical tubes. Then, 4 mL of Lymphoprep (STEMCELL Technologies lnc., Vancouver, BC, Canada) was added to the new tubes, and 8 mL of the diluted blood samples were overlaid on the Lymphoprep. After centrifugation for 20 min at 800× g and at 22 °C, the layer of cells above the Lymphoprep was collected and washed twice with PBS to obtain purified PBMCs that were then suspended in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and transferred to a 1.5 mL tube. The PBMCs were immediately stored at −80 °C until the RNA isolation process.
4
Peripheral Blood Mononuclear Cell Isolation
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Peripheral blood was collected before and after the first and third treatment cycles. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Stemcell Technologies), cryopreserved at local sites and shipped on dry ice to the central laboratory at University of Gothenburg for analysis by flow cytometry. Samples with < 25% viability were excluded from any analyzes.
Leukopacks from healthy blood donors were obtained from Sahlgrenska University Hospital, and PBMCs were isolated by density gradient centrifugation using Lymphoprep (Stemcell Technologies) and cryopreserved in liquid nitrogen. Isolation of NK cells was performed by negative selection using human NK cell isolation kit (Miltenyi Biotec) in accordance with manufacturer’s instructions.
Leukopacks from healthy blood donors were obtained from Sahlgrenska University Hospital, and PBMCs were isolated by density gradient centrifugation using Lymphoprep (Stemcell Technologies) and cryopreserved in liquid nitrogen. Isolation of NK cells was performed by negative selection using human NK cell isolation kit (Miltenyi Biotec) in accordance with manufacturer’s instructions.
5
Isolation of PBMCs, CD14+ Cells, and T Cells
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Buffy coats from venous blood of anonymized healthy donors were purchased from the Blood Transfusion Centre of Slovenia. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Stemcell Technologies, Vancouver, BC, Canada). The cells were washed twice with phosphate-buffered saline (PBS), counted, and used as a source for the immunomagnetic negative isolation of CD14+ cells (EasySep, Stemcell Technologies) or CD4+ naïve T cells (MojoSort, Biolegend, San Diego, CA, USA). The purity of CD14+ and naïve T cells was greater than 85 and 96%, respectively, as determined by flow cytometry. CD3+ T cells were obtained by negative selection using RosetteSep Human T cell enrichment cocktail (Stemcell Technologies) and by density centrifugation using Lymphoprep. The cells were washed twice with PBS. The purity of T cells was greater than 95%, as determined by flow cytometry.
6
Isolation of Peripheral Blood Mononuclear Cells
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Venous blood (10 mL) was sampled from antecubital veins in ethylenediaminetetraacetic acid (EDTA) coated tubes. The EDTA stabilized blood was prepared within 4 h. In a 15 mL centrifuge tube, we added 5 mL LymphoPrep™ (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) and then 10 mL blood carefully on top of the LymphoPrep layer. LymphoPrep™ is a density gradient medium that allows isolation of PBMC through centrifugation. We centrifuged the tube for 30 min at 4 °C at 1000 g with slow acceleration and deceleration. This process allowed following separation (in order from top to bottom): plasma, PBMCs, LymphoPrep, granulocytes, and erythrocytes. We carefully transferred the PBMC layer into a 2 mL tube, centrifuged (5 min at 4 °C at 1000 g), removed the supernatant, added 1.5 mL 4 °C sterile phosphate-buffered saline, centrifuged (5 min at 4 °C at 1000 g), removed the supernatant, and snap-froze the remaining pellet in liquid nitrogen where it was stored until ribonucleic acid (RNA) extraction and analysis. Transportation to RNA extraction was made using dry ice.
7
Porcine PBMC Isolation from Venous Blood
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Swine PBMC from fresh venous blood collected in 5 mM EDTA (final concentration) were isolated by centrifugation on a discontinuous gradient using Lymphoprep™ (STEMCELL™ Technologies, Vancouver, Canada) as previously described [51 (link)]. Briefly, blood with 5 mM EDTA was diluted two-fold in 10mM phosphate-buffered saline (PBS, pH 7.4) containing 20% anti-coagulant citrate dextrose (ACD) and 2% FBS. Thirty-five milliliters of the diluted blood was added to a SepMate™-50 tube (STEMCELL Technologies) containing 15 mL of Lymphoprep™ and the tubes were centrifuged at 1,100 × g for 10 minutes. The buffy coat cells within the supernatant were collected and rinsed with PBS containing 20% ACD and 2% FBS until platelets were removed. Isolated PBMCs were counted, resuspended in a freezing medium containing 10% dimethyl sulfoxide (J.T. Baker, Center Valley, PA, USA) and 90% FBS, and stored in liquid nitrogen until use.
8
Swine PBMC Isolation via Lymphoprep™
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Swine PBMC were isolated from fresh venous blood collected in 5 mM EDTA (final concentration) by centrifugation on a discontinuous gradient using Lymphoprep™ (STEMCELL Technologies, Vancouver, Canada) [52 (link)]. Briefly, blood in 5 mM EDTA was diluted two-fold in 10mM phosphate-buffered saline (PBS, pH 7.4) containing 20% anti-coagulant citrate dextrose (ACD) and 2% FBS. Thirty-five milliliters of the diluted blood was added to a SepMate™-50 tube (STEMCELL Technologies) containing 15 mL of Lymphoprep™. The tubes were centrifuged at 1,100 × g for 10 minutes. The buffy coat cells, at the supernatant cell interface, were collected and pelleted by centrifugation. The cells were resuspended in PBS containing 20% ACD and 2% FBS, and subjected to cycles of centrifugation until platelets were removed. Isolated PBMCs were counted, resuspended in a freezing medium containing 10% dimethyl sulfoxide (J.T. Baker, Center Valley, PA, USA) and 90% FBS, and stored in liquid nitrogen until used.
9
Isolation of PBMCs from Gastric Cancer
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Autologous whole blood from the same gastric cancer patients was processed for the isolation of PBMCs using a Lymphoprep™ (STEMCELL Technologies, Vancouver, Canada) density gradient medium. Following the manufacturer’s protocol, the blood was diluted (50% v/v) in blood wash buffer (1% FCS in DPBS) and added to Lymphoprep™ in a SepMateTM 50-IVD (STEMCELL Technologies) 50 mL tube. The blood was centrifuged at 1200× g for 10 min and the leukocyte-enriched supernatant was diluted 50% v/v with the blood wash and centrifuged at 300 g for 8 min. The supernatant was removed, and the pellet was resuspended in 10 mL of the blood wash, followed by a centrifugation at 120× g for 10 min in order to separate the leukocytes from the platelets. The pellet was either resuspended in freezing medium (45% RPMI, 45% human serum and 10% DMSO) and stored at −80 °C or, used for growing specific immune cells.
10
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.
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.
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