Healthy adult Chinese donors’ heparinized whole blood (n = 10) was mixed with an equal volume of 3% dextran T-500 (Pharmacosmos, Holbaek, Denmark) in 0.9% NaCl and incubated 20 min at room temperature. Aspirated leukocyte-rich plasma and centrifuged 10 min at 1000 rpm, 5 °C. Discarded supernatant and resuspended cell pellet in a volume of 0.9% NaCl equal to the starting volume of blood. Layered 10 ml Ficoll-Hypaque solution (Solarbio, Beijing, China) beneath the cell suspension which ≤40 ml, and centrifuged 40 min at 1400 rpm, 20 °C with no brake. Aspirated the top layer as well as the Ficoll-Hypaque layer, leaving the neutrophil/RBC pellet, and removed residual RBC with red blood cell lysis buffer (TBDScience, Tianjin, China). After washing with Hanks buffered saline solution (Solarbio, Beijing, China), the cells were resuspended and cultured in IMDM (HyClone/Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, HyClone), 100 IU/ml of penicillin G and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) in a cell incubator (5% CO2 at 37 °C)46 . The morphological characteristics and purity of neutrophils were examined with Giemsa staining, immunofluorescence staining and flow cytometry as described below.
Hanks buffered saline solution
Manufactured by Solarbio
Sourced in China
Hanks Balanced Salt Solution (HBSS) is a commonly used buffer solution in cell culture and biological research. It maintains the pH and osmotic balance of cells and tissues. HBSS contains a mixture of inorganic salts, glucose, and phenol red as a pH indicator.
Automatically generated - may contain errors
Lab products found in correlation
Red blood cell lysis buffer, by TBD Science (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Ficoll hypaque solution, by Solarbio (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Dl dithiothreitol, by Merck Group (1 mentions)
2 protocols using hanks buffered saline solution
1
Isolation of Human Neutrophils from Blood
2
Isolation of Intestinal Immune Cells
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A total of 37 preoperative blood samples and 10 postoperative peripheral blood samples were collected. Peripheral blood mononuclear cells were acquired by density gradient centrifugation (800 × g, 30 min, 20°C) using Ficoll-Paque Plus (Amersham; Cytiva).
Fresh intestinal tissue was repeatedly flushed with 0.9% normal saline. Samples were cut into 5-mm2 pieces. The samples were placed in D-Hanks (Hank's Balanced Salt Solution without Ca2+ and Mg2+; Beijing Solarbio Science & Technology Co., Ltd.) containing DL-dithiothreitol, ethylenediaminetetraacetic acid and 2-mercaptoethanol (all from Sigma-Aldrich; Merck KGaA) and incubated for 20 min in a constant temperature water bath at 37°C. Subsequently, the samples were washed with D-Hanks 3–4 times to completely remove any residual reagent. Then, the samples were digested with Hanks buffered saline solution (Beijing Solarbio Science & Technology Co., Ltd.) containing Liberase™ DL Research Grade and DNase I (both from Sigma-Aldrich; Merck KGaA) for 20 min in a constant temperature water bath at 37°C. The filtrate was collected using a cell strainer (Falcon; Thermo Fisher Scientific, Inc.) to separate the tissues and cell suspension, and then the cells were acquired by centrifugation (300 × g, 10 min, 4°C).
Fresh intestinal tissue was repeatedly flushed with 0.9% normal saline. Samples were cut into 5-mm2 pieces. The samples were placed in D-Hanks (Hank's Balanced Salt Solution without Ca2+ and Mg2+; Beijing Solarbio Science & Technology Co., Ltd.) containing DL-dithiothreitol, ethylenediaminetetraacetic acid and 2-mercaptoethanol (all from Sigma-Aldrich; Merck KGaA) and incubated for 20 min in a constant temperature water bath at 37°C. Subsequently, the samples were washed with D-Hanks 3–4 times to completely remove any residual reagent. Then, the samples were digested with Hanks buffered saline solution (Beijing Solarbio Science & Technology Co., Ltd.) containing Liberase™ DL Research Grade and DNase I (both from Sigma-Aldrich; Merck KGaA) for 20 min in a constant temperature water bath at 37°C. The filtrate was collected using a cell strainer (Falcon; Thermo Fisher Scientific, Inc.) to separate the tissues and cell suspension, and then the cells were acquired by centrifugation (300 × g, 10 min, 4°C).
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