The supernatant from digested adipose tissue (SN-AT) was analyzed by sequentially centrifuged. SN-AT was firstly centrifuged at 2000g for 10 min to collect large EVs (lEVs), then the supernatant (SNI) was collected and further centrifuged at 20,000g for 30 min [4 °C, Beckman Avanti J-26S XP centrifuge, JA25.50, polyamide tube (Cat. 357,003)]. The pellet was collected as mixed EVs (mEVs), and the supernatant (SNII) was further ultracentrifuged at 120,000g for 2 h [4 °C, Himac CP 70MX centrifuge, P40ST, Kadj:328.96, polyamide tube (Cat. 332901A)] to collect sEVs. The supernatant (SNIII) was also collected for western blot analysis. The pellets in every centrifugation step were collected and re-suspended in 100μL PBS for further analysis.
J 26s xp
Manufactured by Beckman Coulter
Sourced in United States
The J-26S XP is a high-speed centrifuge designed for general laboratory use. It features a maximum speed of 26,000 rpm and a maximum RCF of 106,800 x g. The centrifuge has a temperature range of -10°C to +40°C and can accommodate a variety of sample types and volumes.
Automatically generated - may contain errors
Lab products found in correlation
Sanderson separation chamber, by Beckman Coulter (2 mentions)
Pancoll human, by PAN Biotech (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Dynabeads untouched human nk cells kit, by Thermo Fisher Scientific (1 mentions)
Lgm 3 medium, by Lonza (1 mentions)
Penicillin streptomycin solution, by PAN Biotech (1 mentions)
Il 15, by R&D Systems (1 mentions)
Interleukin 2 (il 2), by STEMCELL (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Accutase, by STEMCELL (1 mentions)
Foetal bovine serum (fbs), by Biowest (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Anti cd14 mab, by BD (1 mentions)
5 protocols using j 26s xp
1
Isolation and Fractionation of Extracellular Vesicles from Adipose Tissue
2
Isolation and Culture of Human NK Cells
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Anticoagulated citrate dextrose-A-treated blood from healthy donors was purchased from the Regional Centre of Blood Donation and Blood Therapy in Krakow, Poland. Peripheral blood mononuclear cells (PBMC) were isolated by the standard density gradient centrifugation using Pancoll human (Panbiotech, Aidenbach, Germany). Lymphocytes were then separated from PBMC with the AVANTI J-26S XP elutriation system, equipped with the Sanderson separation chamber (Beckman Coulter, Brea, CA, USA), as described previously (24 (link)). NK cells were isolated from leukocytes using MACS technology and a Dynabeads™ Untouched™ Human NK Cells Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), in which NK cells were isolated via negative selection. NK cells were then cultured in LGM-3 medium (Lonza, Basel, Switzerland) supplemented with 5% v/v heat-inactivated human AB serum (Sigma, St. Louis, MO, USA), 500 U/ml IL-2 (STEMCELL Technologies), 140 U/ml IL-15 (R&D Systems, Minneapolis, MN, USA), and penicillin-streptomycin solution (100 U/ml and 100 µg/ml, respectively) (PAN Biotech GmbH, Bayern, Germany). Each experiment was performed using NK cells isolated from a different blood donor.
3
Isolation and Differentiation of Human Monocyte-Derived Macrophages
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Anticoagulated citrate dextrose-A-treated blood from healthy donors was purchased from the Regional Center of Blood Donation and Blood Therapy in Krakow, Poland. Peripheral blood mononuclear cells (PBMC) were isolated by the standard Ficoll/Isopaque (Pharmacia, Sweden) density gradient centrifugation. Monocytes were then separated from PBMC with the AVANTI J-26S XP elutriation system, equipped with the Sanderson separation chamber (Beckman, USA), as described previously (36 (link)). Isolation purity was over 95% as tested by staining with anti-CD14 mAb (BD Biosciences Pharmingen, USA) and flow cytometry analysis (FACSCanto flow cytometer, Becton Dickinson, USA). Cells were washed and resuspended in RPMI 1640 medium (Thermo Fisher Scientific, USA) and kept in an ice bath until used.
Monocytes obtained by elutriation were differentiated for 8-10 days to hMDMs (37 (link)). Three million monocytes per well were seeded in 6-well plates (Ultra-Low Attachment (ULA) Multiple Well Plate, Corning® Costar®, USA) with RPMI medium supplemented with 10% LPS free Foetal Bovine Serum (FBS, Biowest, France). The medium was changed every 48 hours. On days 8-10, cells were detached using ACCUTASE™ (Stemcell Technologies, USA) and the phenotype profile was performed by immunostaining. The macrophages differentiation markers CD68, CD80 and CD163 were analysed (Supplementary Figure 1
Monocytes obtained by elutriation were differentiated for 8-10 days to hMDMs (37 (link)). Three million monocytes per well were seeded in 6-well plates (Ultra-Low Attachment (ULA) Multiple Well Plate, Corning® Costar®, USA) with RPMI medium supplemented with 10% LPS free Foetal Bovine Serum (FBS, Biowest, France). The medium was changed every 48 hours. On days 8-10, cells were detached using ACCUTASE™ (Stemcell Technologies, USA) and the phenotype profile was performed by immunostaining. The macrophages differentiation markers CD68, CD80 and CD163 were analysed (
4
Purification of Recombinant WbDHFR Enzyme
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WbDHFR was expressed at 25°C in LB media with 100 μg/mL ampicillin and induction overnight with IPTG at 0.3 mM. The enzyme was harvested by centrifuging the E. coli mixture at 5,000 rpm for 30 min at 4°C using a JA-10 rotor in a Beckman Avanti J-26S XP centrifuge. The pellet was collected and supernatant discarded. This pellet was then resuspended using equilibration buffer (10 mM imidazole, 20 mM Na2HPO4, 300 mM NaCl, 0.1 mM DTT, at pH 7.4) and soluble protein prepared by sonication of the wet cell paste followed by centrifugation of the mixture using a Sorvall ST16R centrifuge at 5,000 rpm for 30 min at 4°C. The supernatant, rich in soluble WbDHFR, was collected and pellet discarded. His-tagged WbDHFR was purified at pH 7.4 using Ni-NTA resin. The column was washed with 100 mM imidazole wash buffer (100 mM imidazole, 20 mM Na2HPO4, 300 mM NaCl, 0.1 mM DTT, at pH 7.4) before being eluted with 250 mM imidazole elution buffer (250 mM imidazole, 20 mM Na2HPO4, 300 mM NaCl, 0.1 mM DTT, at pH 7.4). Protein was concentrated, and the buffer was exchanged to 20 mM Na2HPO4, 300 mM NaCl, at pH 7.4 and the concentration was determined spectroscopically at 280 nm using the extinction coefficient 25,440 M-1cm-1.
5
Measuring Water Holding Capacity of Gels
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The WHC of gel was measured according to the method (Zhou et al., 2017 (link)). After cutting into small pieces, approximately 3.0 g of gel samples were weighed accurately (M1) and wrapped with two filter papers. Subsequently, gel samples were centrifuged (J‐26sxp; Avanti, Beckman, USA) at 10,000 rpm for 10 min, and weighed again (M2). The WHC was calculated based on the following equation (Eq. (2 )):
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