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.
Lymphoprep solution
Manufactured by STEMCELL
Sourced in Canada
Lymphoprep is a sterile, isotonic density gradient medium used for the isolation of mononuclear cells from whole blood or buffy coat. It has a density of 1.077 g/mL.
Automatically generated - may contain errors
Lab products found in correlation
Sepmate 50 tube, by STEMCELL (3 mentions)
Sepmate tube, by STEMCELL (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Dulbecco s phosphate buffered saline, by Avantor (1 mentions)
Stemspan cd34 expansion supplement 10x, by STEMCELL (1 mentions)
Cd34 microbead kit ultrapure human, by Miltenyi Biotec (1 mentions)
Fix and perm kit, by Thermo Fisher Scientific (1 mentions)
Stemspan sfem 2 medium, by STEMCELL (1 mentions)
Macs ls column, by Miltenyi Biotec (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
K2edta, by BD (1 mentions)
Blood collection tube, by BD (1 mentions)
Rhtnf α, by Thermo Fisher Scientific (1 mentions)
Pge2 elisa kit, by Thermo Fisher Scientific (1 mentions)
Conical tube, by Corning (1 mentions)
Hank s balanced salt solution, by Corning (1 mentions)
Monocyte isolation kit 2, by Miltenyi Biotec (1 mentions)
B cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Cd3 microbeads, by Miltenyi Biotec (1 mentions)
Cd4 cd25 cd127dim regulatory t cells isolation kit 2, by Miltenyi Biotec (1 mentions)
19 protocols using lymphoprep solution
1
PBMC Isolation from Seropositive and Seronegative Subjects
2
Isolation and Expansion of Human CD34+ Cells
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Citric acid (ACS reagent, ≥99.5%), Diethylentriamine (DETA, 99%), L-Glutamine-Penicillin-Streptomycin solution, Dulbecco’s Phosphate Buffered Saline (DPBS), Float-A-Lyzer dialysis devices (100–500 Da), human serum albumin, EDTA, Selenoprotein-AF647 antibodies and sterile filters (200 nm) were obtained from VWR and antibodies against CD45-FITC/CD34-PE, CD34-APC and the FITC Annexin V Apoptosis Detection Kit I were purchased from BD biosciences. Stem SPAN™ SFEM II medium, Stem SPAN™ CD34+ Expansion Supplement (10x) and Lymphoprep™ solution were bought at STEMCELL™ Technologies and microwave reaction vessels were obtained from CEM GmbH. The CD34 MicroBead Kit UltraPure human, MACS LS columns and 30 pre separation filters were purchased from Miltenyi Biotec and the Fix and Perm Kit was bought from Thermo Fisher Scientific. Separation buffer was prepared freshly by supplementing 500 ml DPBS with 1.5 ml 5% human serum albumin and 1.5 ml 50 mM EDTA.
3
PRRSV Viremia and Antibody Evaluation
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For evaluation of viremia and PRRSV specific neutralizing antibody response, 5–7 mL of blood was collected at 0, 14, 26, 42 dpv and 3, 7, 10, 14 dpc. Plasma was separated using K2 EDTA plus blood collection tubes (BD vacutainer, NJ, USA) and preserved at −80 °C until use in the assays. At 26 dpv and 14 dpc, peripheral blood mononuclear cells (PBMC) were isolated from blood collected in EDTA by gradient centrifugation using lymphoprep solution in sepmate tubes (Stem Cell Technologies, Canada) [32 (link)]. PBMC were resuspended in enriched-RPMI (E-RPMI, RPMI containing 10 % FBS, 200 μm HEPES, 1 mM sodium pyruvate, 25 μm 2-ME, 1× Non-Essential Amino Acid, and 1× antibiotic and antifungal). Bronchoalveolar lavage (BAL) fluid was collected during necropsy as described previously [33 ] and aliquots were stored at −80 °C until use in the assays.
4
Assessing PGE2 Production in PBMC
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Buffy coats from anonymous healthy human subjects were courtesy of the Central Blood Bank, Srinakarin Hospital, Khon Kaen, Thailand. Upon arrival, Lymphoprep solution (STEMCELL Technologies Singapore Pte Ltd, Singapore) was used to extract peripheral blood mononuclear cells from the buffy coats as previously described.23,28 Cells with ≥95% viability were used. Purified PBMCs (1 × 105 cells/well) were plated into each well of a black 96-well plate, stimulated with 10 μL of rhTNF-α (Thermofisher, Waltham, MA, USA) at a final concentration of 10 ng/ml for 6 h before irradiation in either continuous or fractionation modes. The negative control was inflamed cells cultured in RPMI-1640 medium and the positive control was cells pre-treated with 50 μg/mL of indomethacin for 24 h before stimulation with rh-TNF-α. Immediately after irradiation was completed, 50 μL of each sample was tested using a PGE2 ELISA kit (Thermofisher, Waltham, MA, USA) following the manufacturers' instructions. Absorbance at excitation and emission wavelengths of 405 and 420 nm, respectively, was measured using the Varioskan Flash microplate reader. Duplicate experiments with two repetitions (total n = 4) were conducted.
5
Plasma Extraction from Whole Blood
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Whole blood was drawn from test subjects and anticoagulated with K2-EDTA. Samples were processed within 4 hours after collection. Blood samples were diluted with an equal volume of 1× Hanks’ balanced salt solution (Corning) and added slowly on top of Lymphoprep solution (15 ml; STEMCELL Technologies) in 50-ml conical tubes (Corning). Samples were centrifuged at 1200g for 10 min at room temperature. The top layer was harvested as plasma and stored at −80°C.
6
Immune Response Monitoring in Kidney Transplant Recipients
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Blood and urine samples were collected from KTRs at baseline, prior to the second vaccine dose, and at two- and three-months post-vaccination. Blood and urine samples were sent to the clinical lab (for serum creatinine and urine protein-to-creatinine ratio quantification), our research laboratories (for DSA, anti-spike antibody and cellular immune assays) and CareDx, Inc. (for ddcfDNA and PBGEP).
Serum and plasma were obtained from peripheral blood by centrifuging for 15 minutes at 2,500 RPM at room temperature then stored in cryogenic tubes at -80°C. PBMCs were isolated using SepMate™-50 tubes (Stemcell technologies) containing Lymphoprep™ solution (Stemcell technologies) then centrifuged at 1,200g for 10 minutes. The PBMC layer was then transferred to a new 50 mL conical tube and centrifuged at 300g for 8 minutes. The supernatant was discarded, and the pelleted cells were resuspended in 10ml of sterile PBS 1x then centrifuged again at 300g for 8 minutes. Pelleted cells were re-suspended in a solution of GemCell™ human serum (GeminiBio) with 10% of dimethyl sulfoxide (100μL for each 1 million PBMCs), aliquoted in cryogenic tubes then stored at -80°C for 1-3 days before being transferred to a liquid nitrogen freezer.
Serum and plasma were obtained from peripheral blood by centrifuging for 15 minutes at 2,500 RPM at room temperature then stored in cryogenic tubes at -80°C. PBMCs were isolated using SepMate™-50 tubes (Stemcell technologies) containing Lymphoprep™ solution (Stemcell technologies) then centrifuged at 1,200g for 10 minutes. The PBMC layer was then transferred to a new 50 mL conical tube and centrifuged at 300g for 8 minutes. The supernatant was discarded, and the pelleted cells were resuspended in 10ml of sterile PBS 1x then centrifuged again at 300g for 8 minutes. Pelleted cells were re-suspended in a solution of GemCell™ human serum (GeminiBio) with 10% of dimethyl sulfoxide (100μL for each 1 million PBMCs), aliquoted in cryogenic tubes then stored at -80°C for 1-3 days before being transferred to a liquid nitrogen freezer.
7
Isolation of Primary Blood Cells
8
Isolation of Immune Cell Subsets from Buffy Coats
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PBMCs were isolated from buffy coats of healthy donors by density gradient centrifugation using Lymphoprep™ solution (07801, STEMCELL Technologies Inc.,Vancouver, BC, Canada). CD14+ monocytes, CD4+ T cells, and CD4+CD25+CD127dim/− Tregs were separated with magnetic-based isolation kits using the autoMACS Pro Separator station (Miltenyi Biotec, Bergish Gladbach, Germany) according to manufacturer’s protocol.
CD14+ MicroBeads (130-096-533, Miltenyi Biotec, Bergish Gladbach, Germany) were used to separate CD14+ monocytes via positive selection, CD4+ T cell Isolation Kit (CD4+ T cell Microbead Cocktail, CD4+ T cell Biotin-Antibody Cocktail, human, Miltenyi Biotec, Bergish Gladbach, Germany) to separate CD4+ T cells via negative selection and CD4+CD25+CD127dim/− regulatory T cells isolation kit II (human, 130-094-775, Miltenyi Biotec, Bergish Gladbach, Germany) to separate Tregs via pre-enrichment of CD4+CD127dim/− and subsequent positive selection of CD4+CD25+CD127dim/−.
CD14+ MicroBeads (130-096-533, Miltenyi Biotec, Bergish Gladbach, Germany) were used to separate CD14+ monocytes via positive selection, CD4+ T cell Isolation Kit (CD4+ T cell Microbead Cocktail, CD4+ T cell Biotin-Antibody Cocktail, human, Miltenyi Biotec, Bergish Gladbach, Germany) to separate CD4+ T cells via negative selection and CD4+CD25+CD127dim/− regulatory T cells isolation kit II (human, 130-094-775, Miltenyi Biotec, Bergish Gladbach, Germany) to separate Tregs via pre-enrichment of CD4+CD127dim/− and subsequent positive selection of CD4+CD25+CD127dim/−.
9
Isolation of Primary Human Monocytes
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Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats using the Lymphoprep solution and the SepMate tubes (both from StemCell Technologies). Red blood cells were removed by incubating cells in red blood cell lysis buffer (Biolegend) for 10 minutes at room temperature. Primary human monocytes were isolated using an EasySep CD14+ Selection Kit (StemCell Technologies) according to manufacturer’s instructions.
10
Isolation of Monocytes from Donor Blood
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Under the IRB approved protocol NCT00001846, peripheral blood mononuclear cells (PBMCs) were obtained from a healthy de-identified blood bank donor (BBD) buffy coat using SepMate-50 tubes (85450, StemCell Technologies, Canada) and Lymphoprep solution (07801, StemCell Technologies, Canada). Cells were washed thoroughly, twice using PBS pH 7.4 (114-058-101, Quality Biological, USA). Monocytes were isolated according to the manufacturer's protocol using either the Pan Monocyte Isolation Kit (130-096-537, MACS Miltenyi Biotec) for the ex vivo experiment or using the EasySep Human Monocyte Isolation Kit (19359, StemCell Technologies) for all in vitro experiments.
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