Peripheral venous blood was drawn from healthy subjects into vacuum tubes containing acid citrate dextrose (BD Biosciences). Samples were centrifuged for 15 minutes at 400 g at room temperature to collect plasma. Packed blood was mixed with an equal volume of 1 × PBS, layered onto 15 mL of lymphocyte separation medium, known as LSM (Lonza), and centrifuged at 800 g (no brake) for 30 minutes at room temperature. PBMCs collected using a pipette were washed with PBS and resuspended in PBS containing 2% v/v FCS. PBMCs were used for initial flow cytometry analysis. The RosetteSep Human B cell enrichment cocktail (Stemcell Technologies) was used for B cell enrichment according to the manufacturer’s instructions. Briefly, whole blood was mixed with RosetteSep cocktail (50 μL/mL of blood sample) and allowed to incubate for 20 minutes at room temperature. The sample was then diluted by adding an equal volume of PBS/2% v/v FCS. After mixing gently, the diluted sample was layered onto LSM and centrifuged at 1200 g (no brake) for 20 minutes at room temperature. The enriched B cell population was collected from the top of the LSM layer, washed in PBS, and resuspended in PBS/2% v/v FCS. The enriched B cell samples were used as starting material for Bmem cell labeling and sorting.
Acid citrate dextrose
Manufactured by BD
Sourced in United States
Acid-citrate-dextrose is a laboratory reagent used as an anticoagulant solution for the collection and storage of blood samples. It prevents the blood from clotting by inhibiting the coagulation cascade.
Automatically generated - may contain errors
Lab products found in correlation
Prostaglandin e1, by Merck Group (3 mentions)
Cellfix, by BD (3 mentions)
Cd61 pe, by BD (3 mentions)
0.105 m buffered sodium citrate, by BD (3 mentions)
Fibrinogen from human plasma oregon green 488 conjugate, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Corning (3 mentions)
Collagen, by Chrono-Log (3 mentions)
Anti human cd62 pe, by BD (2 mentions)
Cd61 fitc, by BD (2 mentions)
Dihydroethidium dhe, by Merck Group (2 mentions)
Thrombin, by Chrono-Log (2 mentions)
Rosettesep human b cell enrichment cocktail, by STEMCELL (1 mentions)
Ficoll, by BD (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Resolvin e1, by Cayman Chemical (1 mentions)
Cd62 pe, by BD (1 mentions)
25 hydroxyvitamin d3, by Merck Group (1 mentions)
Rpmi 1640, by Cytiva (1 mentions)
8 protocols using acid citrate dextrose
1
Isolation and Enrichment of Human B Cells
2
Isolation of Human Peripheral Blood Mononuclear Cells
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Blood samples were collected in tubes containing acid citrate dextrose (BD). Samples were diluted with an equal volume of Dulbecco's Phosphate-Buffered Saline(DPBS) (Gibco) and poured into SepMate tubes (STEMCELL) preloaded with FICOLL (BD) solution. SepMate tubes were spun, and the layer enriched with PBMCs was isolated. Isolated PBMCs were washed with DPBS twice. Cells were counted and resuspended in freezing media. Each vial was placed in a freezing container and transferred later to a liquid nitrogen tank per the manufacturer’s protocol.
3
Transition Period Neutrophil and Metabolite Sampling
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Blood was sampled from the coccygeal blood vessels for neutrophil isolation (~50 mL/cow) and metabolite analysis (~10 mL/cow) at 3 time points over the transition period; day of calving (d 0) and post-calving at wk 1 [1 wk (d 7 ± 1.6)] and wk 4 [4 wk (d 27 ± 1.1)]. Blood for neutrophil isolation was collected in 6 evacuated tubes (BD, Auckland, New Zealand) containing acid citrate dextrose (solution A; trisodium citrate 22.0 g/L, citric acid 8.0 g/L, dextrose 24.5 g/L). The tubes were inverted 8 times and placed immediately on ice until neutrophil isolation, which occurred within 2 h of blood collection. Blood for metabolite analysis was collected in evacuated tubes containing a lithium heparin anticoagulant. Tubes were inverted and placed immediately on ice; within 30 min, blood tubes were centrifuged at 1,500 × g for 12 min at 4°C, and plasma was aspirated and stored at -20°C until assayed.
4
PBMC Isolation from Whole Blood
5
Flow Cytometry Antibody Protocol
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Antibodies for flow cytometry (anti-human CD61/FITC, CD61/PE, CD62/PE), Cellfix, and 0.105 M buffered sodium citrate or acid-citrate-dextrose (ACD) were obtained from Becton-Dickinson (Franklin Lakes, NJ, USA). Bovine serum albumin (BSA), tetrahydrofuran (THF), trimethylammonium diphenylhexatriene (TMA-DPH), diphenylhexatriene (DPH), and prostaglandin E1 were purchased from Sigma (St. Louis, MO, USA). Phosphate buffered saline (PBS) was provided from Corning (New York, NY, USA). Fibrinogen from Human Plasma Oregon Green 488 Conjugate was purchased from Invitrogen (Carlsbad, CA, USA). Resolvin E1 was from Cayman Chemical (Ann Arbor, MI, USA). Collagen was obtained from Chrono-log Co (Havertown, PA, USA). All other chemicals, unless otherwise stated, were supplied by Avantor Performance Materials Poland S.A. (Gliwice, Poland).
6
Platelet Activation Assay Protocol
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Collagen and thrombin were purchased from Chrono-log Co (Havertown, PA, USA). Antibodies for flow cytometry (anti-human CD62/PE, CD61/PE, CD61/FITC), 0.105 M buffered sodium citrate or acid-citrate-dextrose (ACD), and CellFix were from Becton-Dickinson (Franklin Lakes, NJ, USA). Fibrinogen from Human Plasma Oregon Green 488 Conjugate was received from Invitrogen (Carlsbad, CA, USA). Prostaglandin E1, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and dihydroethidium (DHE) were provided by Sigma (St. Louis, MO, USA). Phosphate buffered saline (PBS) was delivered by Corning (New York, NY, USA). All other chemicals, unless otherwise stated, were supplied by Avantor Performance Materials Poland S.A. (Gliwice, Poland).
7
Platelet Activation Assay Protocol
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Collagen and thrombin were purchased from Chrono-log Co (Havertown, PA, USA). Antibodies for flow cytometry (anti-human CD62/PE, CD61/PE, CD61/FITC), 0.105 M buffered sodium citrate or acid-citrate-dextrose (ACD), and CellFix were from Becton-Dickinson (Franklin Lakes, NJ, USA). Fibrinogen from Human Plasma Oregon Green 488 Conjugate was received from Invitrogen (Carlsbad, CA, USA). Prostaglandin E1, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and dihydroethidium (DHE) were provided by Sigma (St. Louis, MO, USA). Phosphate buffered saline (PBS) was delivered by Corning (New York, NY, USA). All other chemicals, unless otherwise stated, were supplied by Avantor Performance Materials Poland S.A. (Gliwice, Poland).
8
Bovine Neutrophil Isolation and Culture
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At 0 and 3 d relative to parturition, peripheral blood was collected from the coccygeal vein using BD Vacutainer blood collection tubes with acid citrate dextrose (Becton Dickinson and Company, Franklin Lakes, NJ) and centrifuged at 1,500 × g for 20 min. Neutrophils were isolated from peripheral blood as previously reported (Merriman et al., 2015) . For neutrophil isolation, the mononuclear cell layer was removed and, to remove erythrocytes, the neutrophil and erythrocyte layer was mixed with 20 mL cold hypotonic buffer (10.6 mM Na 2 HPO 4 , 2.7 mM NaH 2 PO 4 , pH 7.2) for 1 min followed by 10 mL restore solution (10.6 mM Na 2 HPO 4 , 2.7 mM NaH 2 PO 4 , 462 mM NaCl, pH 7.2), centrifuged 10 min at 450 × g, and repeated until erythrocytes were removed.
Neutrophils were cultured in 1 mL of RPMI 1640 (HyClone Laboratories; Logan, UT) that contained 0.42 mM Ca, 10% FBS (HyClone), and 75 ng/mL 25-hydroxyvitamin D 3 (Sigma-Aldrich, St. Louis, MO). Lipopolysaccharide from Serratia marcescens (Sigma-Aldrich) was added to the cultures at a final concentration of 0 or 100 ng/mL of culture media. Neutrophils were cultured in a 5-mL round-bottomed tube at a concentration of 5 × 10 6 cells/mL in a 37°C humidified 5% CO 2 incubator for 6 h.
Neutrophils were cultured in 1 mL of RPMI 1640 (HyClone Laboratories; Logan, UT) that contained 0.42 mM Ca, 10% FBS (HyClone), and 75 ng/mL 25-hydroxyvitamin D 3 (Sigma-Aldrich, St. Louis, MO). Lipopolysaccharide from Serratia marcescens (Sigma-Aldrich) was added to the cultures at a final concentration of 0 or 100 ng/mL of culture media. Neutrophils were cultured in a 5-mL round-bottomed tube at a concentration of 5 × 10 6 cells/mL in a 37°C humidified 5% CO 2 incubator for 6 h.
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