After the endoscopic biopsy, the fresh tissue samples (2–3 mm3) from NPC and NLH patients were rinsed with phosphate-buffered saline (PBS) on ice. Subsequently, each sample was placed into the 500 µL dissociation medium containing 0.5 mg/mL collagenase IV (Sigma) and 1 mg/mL DNAse I (Sigma) in RPMI-1640 (ThermoFisher Scientific). Samples were minced in the dissociation medium on ice and then incubated for 30 min at 37 °C, with manual vortexing every 10 min. Then, 1 mL cold RPMI-1640 containing 10% fetal bovine serum (FBS, ThermoFisher Scientific) was added and each sample was filtered using 70-µm nylon mesh (ThermoFisher Scientific), and subsequently filtered again using 40-µm nylon mesh (ThermoFisher Scientific). The samples were centrifuged at 300×g for 5 min at 4 °C, and the supernatant was discarded. The single cells were resuspended in 1 mL ACK lysis buffer (ThermoFisher Scientific) and incubated for 5 min. 5 mL cold RPMI-1640 containing 10% FBS was added and the cell mixture was centrifuged at 300×g for 5 min at 4 °C. The single-cell pellet was resuspended in PBS without calcium and magnesium ions to reach the density ≤1000 cells/µL.
70 m nylon mesh
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 70 µm nylon mesh is a laboratory filtration product. It has a pore size of 70 micrometers and is made of nylon material.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
40 m nylon mesh, by Thermo Fisher Scientific (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
M199 media, by Thermo Fisher Scientific (1 mentions)
2 ml tube, by Corning (1 mentions)
Dnase 1, by Roche (1 mentions)
Collagenase p, by Roche (1 mentions)
M csf, by Miltenyi Biotec (1 mentions)
Picopure rna extraction kit, by Arcturus (1 mentions)
5 protocols using 70 m nylon mesh
1
Nasopharyngeal Carcinoma Cell Isolation
2
Isolation and Characterization of ADSCs
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ADSCs were isolated from VAT of 6-wk-old WT, K43M, R31C, and K43M;Runx1fl/fl;CRE- mice and grown in vitro (Hou et al., 2018 (link)). Epididymal white adipose tissue (eWAT) from male mice and gonadal white adipose tissue (gWAT) from female mice were used in this study. ADSCs were isolated from the adipose stromal vascular fraction (SVF) according to a published procedure (Permana et al., 2004 (link)). Briefly, freshly collected eWAT and gWAT from normal chow-fed animals were digested in 1 x HBSS containing 1 mg/mL type I collagenase (Millipore Sigma, United States) at 37°C for 1 h. The suspension was filtered through a sterile 100 µm nylon mesh (Thermofisher Scientific) and centrifuged at 1,000 rpm for 5 min. The pellet fraction containing pre-adipocytes was washed 2 times with PBS +2% FBS and then incubated with red blood cell lysis buffer to remove red blood cells. Cells were then filtered through a sterile 70 µm nylon mesh (Thermofisher Scientific) and centrifuged at 1,000 rpm for 5 min and counted. Fractions of 3×105 cells were used for subsequent staining and analysis using flow cytometry.
3
Dissociation and Live Cell Staining
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Biopsy or metastatic tumour were dissected and transferred to a 2 ml tube (Axygen, China), each containing 1 ml prewarmed M199 media (Thermo Fisher Scientific, USA), 2 mg/ml collagenase P (Roche, USA) and 10 U/µl DNase I (Roche, USA) as described by Tirosh et al.17 (link). Tissues were digested for 60 min at 37 °C and then pipetted up and down every ten times every 10 min. The tissue suspensions were then filtered with a 70 µm nylon mesh (Thermo Fisher Scientific, USA) and centrifuged at 450g for 5 min. Pellets were resuspended for live cell staining using CFSE incubation for 5 min.
4
Generation of Mouse Bone Marrow-Derived Macrophages
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Mouse bone marrow-derived macrophages (BMMs) were generated following the method previously published using 8–12 week-old C57Bl/6 mice [28 (link)]. Briefly, bone marrow material was obtained by flushing mouse femurs and tibias in complete RPMI. The cell suspensions were filtered through a 70 µm-nylon mesh (ThermoFisher, Waltham, MA, USA) and then centrifuged at 400 rpm for 5 min. Then, ammonium–chloride–potassium (ACK) lysis buffer was used to remove red blood cells. The remaining cells were incubated in untreated 100 mm × 15 mm Petri dishes for 7 days in the presence of complete RPMI supplemented with 30 ng/mL of M-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany). Fresh medium was added after 3 days of culture.
5
Isolating Fluorescent Oenocytes from Mosquitoes
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An. gambiae mosquitoes were reared at 28°C under 80% humidity and at a 12/12 h day/night cycle. Larvae were fed with fish food (TetraMin, Tetra GmbH), and adult mosquitoes were fed ad libitum with 10% sugar. To generate mosquitoes with fluorescent oenocytes we crossed males from the UAS-mCD8: mCherry responder line (Adolfi et al., 2018 (link)) with virgin females of the oeno-Gal4 driver line (Lynd et al., 2019 (link)). Adult progeny (2–4 days old) were collected, anesthetised on ice and dissected in 1X PBS. The head, thorax and internal tissues (midgut, malpigian tubules and reproductive tissues) were removed and the remaining integument (carcass) was cut open. Each sample (N = 12 in total, Supplementary file 5 ) consisted of 30 carcasses. Samples were washed twice with 1X PBS and incubated for 30 min at 37°C with 0,25% trypsin in 1X PBS. After incubation tissues were washed twice with 1X PBS and homogenised by pipetting up and down in 1X PBS containing 1% fetal bovine serum. Dissociated cells were filtered through a plastic filter mesh (ThermoFisher 70 µm Nylon Mesh). For samples used to isolate oenocytes (N = 6), cells were immediately used for FACS sorting. In the case of total carcass cells (N = 6) total RNA was extracted after filtering using the Arcturus PicoPure RNA extraction kit.
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