In the alkaline comet assay human lymphocytes were used. They were isolated from peripheral blood obtained by the finger prick method. To 1 mL of ice cold 1× PBS, 40 μL of peripheral blood was added and allowed to stand on ice for 30 min. Lymphocytes were separated from whole blood samples by standard centrifugation with 100 μL of the Histopaque medium (Sigma-Aldrich). After the centrifugation, 200 μL of isolated cells were resuspended in 1 mL of 1× PBS buffer and re-centrifuged.
Histopaque medium
Manufactured by Merck Group
Sourced in Germany, United States
Histopaque medium is a density gradient solution used for the isolation and purification of mononuclear cells from blood or bone marrow samples. It facilitates the separation of different cell types based on their density differences.
Automatically generated - may contain errors
Lab products found in correlation
Red blood cell lysing buffer, by Merck Group (1 mentions)
Cell free dna bct tube, by Streck (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Freund s incomplete adjuvant, by Statens Serum Institut (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
4 protocols using histopaque medium
1
Lymphocyte Isolation for Comet Assay
2
Isolation of Lung Neutrophils Using FACS Aria
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In order to recover lung neutrophils using the "FACS aria", mice (n = 6) received intra-tracheal instillations of recombinant CCL11 (10 ng) on J0, J2, J5, and J7 and were sacrificed on J8. Lungs were incubated for 45 min with shaking at 37 °C in medium without serum and IV collagenase (1 mg/ml) (Gibco, Belgium). After homogenization, FBS was added in equal volume to neutralize collagenase IV. Samples were centrifuged at 335 g for 5 min and treated by addition of "Red Blood Cell Lysing Buffer" (Sigma, Germany). The samples were then separated into several phases on Histopaque® medium (Sigma, Germany). The granulocyte phase was stored and washed in PBS 2% FBS. Neutrophils ("high" SSC; "low" FSC) were labeled with antibodies (CD45+, CD11b+, Gr1+, CCR3+). On the basis of these markers, cells were isolated using the "FACS Aria with a purity of 93.92%. Once recovered, the cells were fixed on a slide (CytoSpin; Statspin Cytofuge 2; Iris, USA) to confirm they are neutrophils.
3
Isolation of human PBMCs
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Human peripheral blood mononuclear cells (PBMCs) were obtained from three healthy donors with approval of the Institutional Review Board (IRB) of Yonsei University (7001988-202106-HR-864-04). Blood from healthy donors was collected in Cell-Free DNA BCT tubes (STRECK). PBMCs were isolated according to standard procedures by density gradient centrifugation using Histopaque medium (Sigma, USA) and washed with PBS (Gibco, USA). To reduce platelet contamination, five additional low-speed centrifugation (10 min at 120 × g) steps were performed with the centrifuge brake off. Cells were resuspended in PBS-BSA and counted for subsequent use in experiments.
4
Llama In-vivo Immunization Protocol
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In-vivo immunization of llama was performed following the protocol described by Pardon and co-workers with some modifications (Pardon et al., 2014 (link)). Briefly, 200 μg of rec-NadA was used for the first immunization followed by 5–weekly immunizations with 100 μg of the same protein (total 700 μg of protein used for immunization). For the first immunization, rec-NadA was mixed with Freund's complete adjuvant (1:1) (Sigma-Aldrich) and 1 mL of the mix was injected i/m in quadriceps femoris. Subsequent immunizations were performed using the emulsion of rec-NadA and Freund's incomplete adjuvant (1:1) (Statens serum institut, Copenhagen, Denmark). One week after the last immunization, 100 mL of blood was collected from the jugular vein in the presence of heparin (2500 IU) (Zentiva a.s., Czech Republic). Peripheral blood mononuclear cells (PBMC) were isolated from the heparinized blood using density gradient centrifugation in Histopaque medium (Sigma-Aldrich) as described in the manufacturer's instruction. RNA was isolated from the PBMC using the RNeasy mini kit (Qiagen) and reverse-transcribed with RevertAid (Thermo Scientific, Bratislava, Slovakia) following the manufacturer's instructions. sdAb-Not-R primer (Table 1 ) was used in the reverse transcription reaction.
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