IVDs were harvested from the lumbar spines of C57BL/6 mice immediately after they were euthanized according to the IACUC animal protocol approved by Peking University Third Hospital Ethics Committee (No. A2019021). The NP tissues were carefully separated from the annulus fibrosus under the microscope and cut into small fragments. Fragments were digested with 0.25% Type II collagenase (Sigma) for 1 h, followed by trypsin–EDTA solution (0.2% trypsin, 1 mM EDTA, Gibco) for 5 min, respectively. Then the isolated NP cells were cultured at 37°C under 5% CO2 conditions in Dulbecco's modified Eagle medium (DMEM, HyClone, Thermo Scientific, Logan, UT, USA) containing 20% foetal bovine serum (FBS, Gibco, Grand Island, NY, USA). When NP cells reached 80%–90% confluency, they were passaged with trypsin–EDTA solution (0.25% trypsin, 1 mM EDTA, Gibco). The third passage (P3) NP cells were applied for the subsequent experiments.
Ethylene diamine tetraacetic acid (edta)
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Switzerland, France, China, Japan, Canada, Belgium, Italy, Spain, India, Sweden, Netherlands, Australia
EDTA is a chemical compound used as an anticoagulant in laboratory settings. Its primary function is to prevent the coagulation or clotting of blood samples, which is essential for various analytical and diagnostic procedures.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Thermo Fisher Scientific (366 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (207 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (118 mentions)
Penicillin, by Thermo Fisher Scientific (101 mentions)
Streptomycin, by Thermo Fisher Scientific (101 mentions)
Bovine serum albumin (bsa), by Merck Group (84 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (69 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (68 mentions)
Hepes, by Thermo Fisher Scientific (56 mentions)
Dithiothreitol (dtt), by Merck Group (43 mentions)
L glutamine, by Thermo Fisher Scientific (39 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (39 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (36 mentions)
Matrigel, by Corning (36 mentions)
Rpmi 1640, by Thermo Fisher Scientific (36 mentions)
Dnase 1, by Merck Group (35 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (30 mentions)
Dmem f12, by Thermo Fisher Scientific (28 mentions)
Geltrex, by Thermo Fisher Scientific (26 mentions)
Glutamax, by Thermo Fisher Scientific (24 mentions)
1 711 protocols using ethylene diamine tetraacetic acid (edta)
1
Mouse Intervertebral Disc Cell Isolation
2
Isolation of Mononuclear Cells from Whole Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was collected from the jugular vein with a syringe containing heparin. Mononuclear cells were isolated using density gradient centrifugation with Histopaque®-1083 (Sigma-Aldrich). Blood was diluted at a one to one ratio with elution media (58.4 mM sucrose (Sigma-Aldrich), 10 ml 5 mM EDTA (Invitrogen), 100 mL 10x PBS (Gibco), 900 mL Milli-Q Water) at room temperature and carefully layered on top of the Histopague in a conical tube. The layered blood is centrifuged at 9000 RCF for 30 min. Lymphocytes are collected by harvesting the buffy coat. Blood and lymph-borne cells were washed with wash media (RPMI 1640 medium with GlutaMAX™ (Gibco®), 0.2% BSA (Sigma-Aldrich), and 25 mM HEPES (Gibco®)), and, when necessary, red blood cells were lysed using red blood cell lysing buffer (155 mM ammonium chloride (Sigma-Aldrich), 10 mM sodium bicarbonate (Sigma-Aldrich), and 0.1 mM EDTA (Gibco)). Isolated cells were resuspended in wash media, counted by hemocytometer, and kept on ice until staining.
3
hESC and iPSC Maintenance and Passaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hESCs and iPSCs were maintained in iPS brew medium (StemMACS iPS-Brew XF and 0.5% penicillin/streptomycin (Gibco)) on Nunc Δ multidishes coated with LN521 (0.7 μg/cm2; Biolamina). Cells were passaged approximately 1:2–1:10 every 2–5 days, starting with one rinse with DPBS (Gibco) followed by dissociation using 0.5 mM EDTA (75 μl/cm2; Gibco) at 37 °C for 7 min. Following incubation, EDTA was carefully removed from the well and the cells were washed off and collected in 10 ml wash medium (9.5 ml DMEM/F-12 (31330–038; Gibco) with 0.5 ml knockout serum replacement (Gibco)). The cells were then centrifuged at 400 × g for 5 min, supernatant aspirated, and the cells were resuspended in iPS brew medium supplemented with 10 μM Y27632 (Rock inhibitor; Miltenyi) and plated for expansion. The media was changed daily to fresh iPS brew medium.
4
Intestinal Organoid-Macrophage Coculture to Study IBD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh mouse and human intestinal tissues were incubated with 2 mmol/L EDTA (Gibco, USA) for 30 min at 4 °C and 5 mmol/L EDTA (Gibco, USA) for 30 min (×2) at 4 °C on a roller. The tissues were then filtered and 10 mL of 0.1% BSA was added (Solarbio, China). Next, the isolated crypt units were plated to a density of 200 crypts per well in a 40-μL droplet of Matrigel mixed with IntestiCult media. Crypt-containing Matrigel droplets were overlaid with IntestiCult complete organoid media (Stemcell Technologies, Vancouver, BC) supplemented with penicillin/streptomycin. BMDMs were then plated on the top membrane of transwell inserts for a minimum incubation of 12 h with LPS (100 ng/mL) stimulation. When a cell attachment of 60% was achieved, the BMDMs were cocultured with colonic organoids in the presence of LPS (100 ng/mL, refresh every 24 h) to investigate the effect of BMDMs on the progression of IBD in vitro.
5
Isolation of Peripheral Blood Mononuclear Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS solution (Sigma-Aldrich). Fresh peripheral blood was collected before the surgery in EDTA anticoagulant tubes. Briefly 8 ml of fresh peripheral blood were resuspended in 1X PBS (Invitrogen) with 1% FBS and 2mM EDTA (Gibco) and the layered onto Ficoll-Paque PLUS solution. Subsequently cells are centrifuged for 20 minutes at room temperature at 2000 rpm without breaks. After centrifugation tumor-infiltrating lymphocytes were carefully transferred to a new tube and washed with 1X PBS with 1% FBS and 2mM EDTA (Gibco).
6
Differentiation of Mouse Bone Marrow-Derived DCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse bone marrow derived DCs were differentiated from bone marrow isolated from mouse tibiae. 20 million cells were seeded in gamma-irradiated heavy 14 cm dishes (Greiner Bio-One) in 20ml of BMDCs (bone marrow-derived DCs) medium composed by IMDM, 10% FBS, PenStrep (GIBCO), 50μM β-mercaptonethanol (GIBCO), and granulocyte-macrophage colony stimulating factor (50 ng/mL)-containing supernatant obtained from transfected J558 cells. Cells were split at day 4 and day 7 and harvested at day 10. At day 4 the supernatant was recovered, and the adherent cell were recovered by incubating the dishes in 6ml of PBS (GIBCO) containing 5mM EDTA (GIBCO). Cells were counted and reseeded in BMDCs medium at a concentration of 0.5 million cells per ml, 20ml per 14cm dish. At day 7 the culture supernatant was gently discarded and the cells were recovered by incubating the dishes in 6ml of PBS containing 5mM EDTA (GIBCO). Cells were counted and reseeded in BMDCs medium at a concentration of 0.5 million cells per ml, 20ml per 14cm dish. At day 10, the culture supernatant was gently discarded and semi-adherent cells were recovered by extensive flushing of the dishes with 10ml of pre-warmed BMDCs medium. The cells were counted and used for further applications. Number of mice and experimental replicates are indicated in the respective figure legend.
7
Induced Pluripotent Stem Cell Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human iPSCs were obtained as reported previously (Cui et al., 2020 (link)), which were reprogrammed by an inactivated Sendai virus from healthy female peripheral blood mononuclear cells, and cultured in mTesR1 complete medium (STEM CELL Technologies, BC, Canada) on diluted Matrigel (1:100, Corning, NY, United States). The cells were subcultured every 4 days by incubation with 0.5 mM EDTA (Thermo Fisher Scientific, United States) at 37°C for 3 min. Human beta cells were cultured in high glucose DMEM supplemented with 10% serum replacement. At 80% confluence, the cells were trypsinized with 0.25% trypsin and 0.01% EDTA (Gibco).
8
Isolation of Peripheral Blood Mononuclear Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS solution (Sigma-Aldrich). Fresh peripheral blood was collected before the surgery in EDTA anticoagulant tubes. Briefly 8 ml of fresh peripheral blood were resuspended in 1X PBS (Invitrogen) with 1% FBS and 2mM EDTA (Gibco) and the layered onto Ficoll-Paque PLUS solution. Subsequently cells are centrifuged for 20 minutes at room temperature at 2000 rpm without breaks. After centrifugation tumor-infiltrating lymphocytes were carefully transferred to a new tube and washed with 1X PBS with 1% FBS and 2mM EDTA (Gibco).
9
Human and Chimpanzee iPSC Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used two human iPSC lines generated by mRNA transfection (RBRC-HPS0328 606A1 and RBRC-HPS0360 648A1, both from RIKEN; from here on referred to as HS1 and HS2, respectively). We used two chimpanzee iPSC lines: one generated by mRNA transfection (Sandra A, herein referred to as PT1) and the other with viral vector transduction (PR00818 PTCL-5, herein referred to as PT2) (Marchetto et al., 2013; Mora-Bermu ´dez et al., 2016) . iPSCs were maintained on LN521-coated (0.7 mg/cm 2 ; Biolamina) Nunc multidishes in iPS media (StemMACS iPS-Brew XF and 0.5% penicillin/streptomycin (GIBCO)). Cells were passaged 1:2-1:6 every 2-4 days by being rinsed once with DPBS (GIBCO) and dissociated using 0.5 mM EDTA (75 ml/cm 2 ; GIBCO) at 37 C for 7 minutes. Following incubation, EDTA was carefully aspirated from the well and the cells were washed off from the dish using washing medium (9.5 mL DMEM/F-12 (31330-038; GIBCO) and 0.5 mL knockout serum replacement (GIBCO)). The cells were then centrifuged at 400g for 5 minutes and resuspended in iPS brew medium supplemented with 10 mM Y27632 (Rock inhibitor; Miltenyi) for expansion. The media was changed daily (Grassi et al., 2019; Nolbrant et al., 2017) .
10
Isolation and Characterization of Dental Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DPSCs were obtained from pulp tissue of incisors and PDLSC from periodontal ligament of molars from 6-weeks old male Wister Hann rats (weight 120g; Charles River Laboratories, via Aston University, Birmingham, UK) as described previously (9, 19, 20) . In brief, to isolate DPSC, pulp tissue was minced into pieces of ~1 mm 3 and digested at 37 °C with 0.25 % (w/v) trypsin and 1 mM EDTA (Gibco, Paisley, UK) for 30 min. After centrifugation at 900g for 5 min, the cell pellet was re-suspended and seeded in αMEM/20 % FBS. For PDSLC isolation, the extracted periodontal tissue was incubated with 0.25 % (w/v) trypsin and 1 mM EDTA (Gibco, Paisley, UK) for 45 min at 37 °C. Cell pellets were re-suspended and seeded in αMEM/20 % FBS. The established DPSC and PDLSC cultures, used for the experiments at passages 3-5, were shown to express a range of stem cell markers and had multilineage differentiation potential (9) .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!