Conical tube

Manufactured by BD

Sourced in United States

Conical tubes are commonly used laboratory containers. They are designed with a tapered, cone-shaped bottom to facilitate sample collection and processing. Conical tubes are available in various sizes and materials to accommodate different volume requirements and sample types.

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15 ml conical tube Falcon conical tube Polypropylene conical tubes 50 mL conical tubes Sterile conical polypropylene tube with flat-top screw cap Polystyrene conical centrifuge tube Falcon™ conical centrifuge tubes Conical shaped polypropylene tube Conical bottom tubes 15 mL conical centrifuge tubes

30 protocols using conical tube

Controlled Release of BMP-2 from Fiber Scaffolds

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The release profiles of BMP-2 from the BMP-2/PCL fibers and BMP-2/AAc-PCL fibers were evaluated by an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. In brief, BMP-2/PCL fibers and BMP-2/AAc-PCL fibers were placed into a 50 mL conical tube (Falcon, USA) containing 1 mL PBS buffer (pH 7.4) with gentle shaking at 100 rpm and 37°C. At predesignated time intervals of 1, 3, 5, and 10 hr, and 1, 3, 5, 7, 14, 21, and 28 days, PBS was extracted and collected from the specimens and fresh PBS was replaced into the 50 mL conical tube. The amount of BMP-2 released was determined by an ELISA kit using a microplate reader at 450 nm.

Yun Y.P., Lee J.Y., Jeong W.J., Park K., Kim H.J., Song J.J., Kim S.E, & Song H.R. (2015). Improving Osteogenesis Activity on BMP-2-Immobilized PCL Fibers Modified by the γ-Ray Irradiation Technique. BioMed Research International, 2015, 302820.

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Brain Microvascular Isolation and Analysis

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Brain microvessels were isolated from one cerebral cortex as previously described, with minor modifications.⁴ Briefly, PND 14 (juvenile) offspring were euthanized under isofluorane anesthetic by cardiac puncture and exsanguination followed by decapitation. Brains were removed and one cerebral cortex was isolated for primary BEC culture, the other stabilized in RNAlater™ (Invitrogen, MA, USA) for mRNA expression analysis. The cerebral cortex was dissected into small pieces using surgical scissors in a 50 ml conical tube (Falcon) with 10 ml of RNAlater™ Stabilization Solution (Invitrogen, MA, USA) on ice, and stored overnight at 4°C, for qPCR analysis or homogenized (Potter‐Evehljem Tissue Grinder; Sigma, MA, USA), resuspended in dextran (Millipore Sigma, MA, USA) solution [17.5% (wt/vol) in Hank's Balanced Salt Solution (HBSS, Wisent BioProducts, Quebec, Canada)] and centrifuged (3000 g, 30 min, 4°C) for microvessel isolation. After centrifugation, the brain parenchyma and dextran were aspirated, leaving the microvessel pellet. The microvessel pellet was resuspended into 3 ml of HBSS, aliquoted into three 2 ml Eppendorf tubes, and spun on a benchtop centrifuge (10 000 rpm, 10 min, 4°C). The HBSS was removed, and microvessel pellets were stored at −80°C for downstream applications.

Eng M.E., Bloise E, & Matthews S.G. (2022). Fetal glucocorticoid exposure leads to sex‐specific changes in drug‐transporter function at the blood‐brain barrier in juvenile guinea pigs. The FASEB Journal, 36(4), e22245.

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Fluorescent Exosome Labeling and Uptake

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Exosomes were labeled by carbocyanine dye DiO (Thermo Fisher Scientific) following a previously reported protocol [39 (link)]. Briefly, 10 μg/mL exosomes were incubated with DiO for 20 min at 37°C followed by PBS washing three times. 50,000 DPSCs were seeded on glass cover slides and cultured in 24-well-plates. Labeled exosomes were added to the culture media of cells and incubated for 30 minutes. Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA). Immunofluorescence and confocal microscopy are described in detail below. To evaluate the uptake of exosomes released from EXO-MS polymer spheres, DiO labelled exosomes were used in the fabrication of EXO-MS, following the described particle fabrication protocol presented herein. Following lyophilization and sterilization, particles were incubated in PBS for two weeks, changing PBS every 48 hours, then with cell culture media for two hours on a shaker at 37°C. Particles were allowed to settle to the bottom of a 15-mL conical tube (Falcon), and supernatant conditioned media, containing released fluorescently labelled exosomes, was collected and incubated with cells in the same method described above, for 30 minutes. Their uptake is visualized by confocal microscopy.

Swanson W.B., Gong T., Zhang Z., Eberle M., Niemann D., Dong R., Rambhia K.J, & Ma P.X. (2020). Controlled Release of Odontogenic Exosomes from a Biodegradable Vehicle Mediates Dentinogenesis as a Novel Biomimetic Pulp Capping Therapy. Journal of controlled release : official journal of the Controlled Release Society, 324, 679-694.

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Chondrogenic Pellet Culture Protocol

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Pellet culture was performed by spinning down 3×10⁵ cultured cells in each 15ml conical tube (BD Falcon) which were then grown in serum-free DMEM containing an insulin-transferrin-selenious acid mix (ITS) (Becton-Dickinson), 50 μg/ml L-ascorbic acid 2-phosphate (WAKO), 100 μg/ml sodium pyruvate, 40 μg/ml L-proline (both from Invitrogen, Life Technologies), 0.1 μM dexamethasone (Sigma-Aldrich) and 10 ng/ml transforming growth factor β1 (TGF-β1; Peprotech) (13 (link)). Three weeks later, pellets were fixed in 10% formalin, dehydrated in ethanol, and embedded in paraffin. Sections (5 μm thick) were rehydrated and stained with Alcian blue and nuclear fast red for the detection of sulfated glycosaminoglycans and nuclei, respectively.

Chen W.C., Baily J.E., Corselli M., Diaz M., Sun B., Xiang G., Gray G.A., Huard J, & Péault B. (2015). Human Myocardial Pericytes: Multipotent Mesodermal Precursors Exhibiting Cardiac Specificity. Stem cells (Dayton, Ohio), 33(2), 557-573.

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Isolation and Transfer of Intestinal IELs

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Colon tissue was harvested and flushed with PBS (Gibco) before being finely minced and placed in a 50 ml conical tube (Falcon) with 10 ml of PBS supplemented with 1 mM EDTA and 7.5 mM HEPES. Tissue was shaken at max speed in a tabletop vortex for 10 minutes to disassociate the epithelial fraction. The epithelial fraction was passed through a 70um cell strainer (VWR) before proceeding to magnetic enrichment. IELs were purified using a Easysep negative selection magnetic enrichment kit (STEMCELL), supplemented with 1ul/sample biotin conjugated anti-EPCAM (clone G8.8). Isolated cells were introduced to Rag1^-/- hosts by tail vein injection. Homeostatic T cell proliferation in recipient animals was monitored weekly by cheek bleed and flow cytometry until circulating CD3+ lymphocyte levels were stable (10 weeks).

Lefferts A.R., Norman E., Claypool D.J., Kantheti U, & Kuhn K.A. (2022). Cytokine competent gut-joint migratory T Cells contribute to inflammation in the joint. Frontiers in Immunology, 13, 932393.

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Fabrication of RGD-functionalized PEG Microgels

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PEG-4MAL (20 kDa, Laysan Bio) was dissolved in phosphate-buffered saline (PBS) at 5% (w/v) then filtered through a 40 μm cell strainer (Corning). For experiments involving the injection of microgels in vivo, microgels were functionalized with GRGDSPC (RGD, Genescript). PEG-4MAL was reacted with 2.0 mM RGD for 30 minutes at 37 °C to create RGD-functionalized macromer. For all other experiments, RGD was not used in the formation of microgels. Crosslinker solutions (DTT (Sigma) or GCRDVPMSMRGGDRCG (VPM, Genescript) or combinations of both) were prepared at predetermined molar concentrations and then adjusted to a pH of 4.5 to slow down gelation kinetics in order to prevent the device from clogging. PEG-4MAL and crosslinker were then infused into the flow-focusing microfluidic device to form polymer droplets. Droplets were formed within an oil solution consisting of light mineral oil (Sigma) mixed with 2% SPAN80 (Sigma) and then collected into a 15 mL conical tube (Falcon). After formation, microgels were washed in PBS five times by centrifugation to remove mineral oil and surfactant.

Foster G.A., Headen D.M., González-García C., Salmerón-Sánchez M., Shirwan H, & García A.J. (2016). Protease-Degradable Microgels for Protein Delivery for Vascularization. Biomaterials, 113, 170-175.

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Isolation and Characterization of Ovarian Granulosa-like Stem Cells

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Singly dissociated ovarian cells were suspended in 200 µL DPBS and loaded onto the top of a discontinuous 7-step Percoll (Sigma-Aldrich, St. Louis, MO, USA) solution consisting of 1 mL each of 20%, 25%, 30%, 35%, 40%, 50%, and 60% in a 15 mL conical tube (Falcon, Durham, NC, USA). Each Percoll solution was prepared by the manufacturer’s instruction. After centrifugation at 800× g for 30 min, the cells from each density fraction were carefully harvested, washed twice with DPBS, and then used for experiments. Cell morphologies were observed under a phase-contrast inverted microscope (TS-100F, Nikon, Tokyo, Japan). Morphology of OGSCs was defined as cells harboring a large nucleus with one or two prominent nucleoli [24 (link),25 (link)].

Ryu J.H, & Gong S.P. (2020). Enhanced Enrichment of Medaka Ovarian Germline Stem Cells by a Combination of Density Gradient Centrifugation and Differential Plating. Biomolecules, 10(11), 1477.

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Comprehensive Cell Extraction from Cytobrushes

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The two cytobrushes were processed sequentially and the extracted cells were combined for staining and flow cytometric analysis. Cytobrushes were inserted into 25 mL serological pipettes containing 20 mL room temperature phosphate buffered saline (PBS). The brush was moved in and out of the tip of the pipette while the PBS was gradually expelled, extracting the cells from the cytobrush and washing them through a 100 µm cell strainer into a 50 mL conical tube (BD, Franklin Lakes, NJ, USA). This was repeated with another 10 mL PBS and then the cytobrushes were scraped on the edge of the cell strainer and discarded. The 5 mL RPMI-1640 in the transport tube was passed through the strainer and the tube washed with an additional 15 mL PBS. If the strainer became clogged with mucus, the mucus was pipetted up and down in the serological pipette and broken up. After straining, the 50 mL cell suspension was pipetted up and down several times to break up any remaining mucus. The full cytobrush processing protocol is included in File S2.

McKinnon L.R., Hughes S.M., De Rosa S.C., Martinson J.A., Plants J., Brady K.E., Gumbi P.P., Adams D.J., Vojtech L., Galloway C.G., Fialkow M., Lentz G., Gao D., Shu Z., Nyanga B., Izulla P., Kimani J., Kimwaki S., Bere A., Moodie Z., Landay A.L., Passmore J.A., Kaul R., Novak R.M., McElrath M.J, & Hladik F. (2014). Optimizing Viable Leukocyte Sampling from the Female Genital Tract for Clinical Trials: An International Multi-Site Study. PLoS ONE, 9(1), e85675.

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Robust RPE Differentiation from hiPSCs

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All reagents were purchased from Invitrogen (Carlsbad, CA) unless noted otherwise. The procedure for differentiating human iPS cells toward RPE was performed as previously described [17 ]. Briefly, when the iPS culture reached 60–70% confluency, individual colonies were lifted using accutase (Stem Cell Technologies). Colonies were allowed to settle in a 15 mL conical tube (BD), and, after media was replaced with mTeSR 1 media, were transferred to a T25 flask (Corning) to initiate differentiation (day 0). Over the following 4 days, the colonies were gradually transitioned to neural induction medium (NIM) which consisted of DMEM/F12, 1% N2 supplement, MEM non-essential amino acids, and 2 μg/mL heparin. At day 6, the colonies were transferred to a 10-cm dish coated with laminin in NIM, changed every 2 days. Rosettes were removed from the culture by light trituration at day 16. The remaining cells were switched to retinal differentiation medium (DMEM/F12 (3:1), 2% B27 without retinoic acid, and 1% antibiotic/antimycotic). This culture was maintained in RDM until day 80 for dissection and passaging, which was performed as described. This process was performed 3 times to generate the 3 technical replicates used for this study.

Au E.D., Fernandez-Godino R., Kaczynksi T.J., Sousa M.E, & Farkas M.H. (2017). Characterization of lincRNA expression in the human retinal pigment epithelium and differentiated induced pluripotent stem cells. PLoS ONE, 12(8), e0183939.

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Sucrose Preference in Mice: Juvenile and Adult

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Preference for a 2% w/v sucrose solution was assessed in the home cage. Two 50 mL conical tubes (Falcon) with neoprene stopper and straight sipper tubes containing either 2% sucrose solution or plain tap water were introduced to the cage. The mice were group housed for juvenile testing on PND 42 and one cage of two to three animals were represented as a single data point. The mice were given a habituating day on PND 41 before being tested on PND 42. On both days the two bottles were available for 4 h beginning at the onset of the dark phase. The bottles were weighed before and after to calculate the amount of sucrose and water consumed. The sucrose preference was measured as the ratio of sucrose solution consumption over total liquid consumption. Since PND 60 mice from the same cage received different injections, for adult testing the mice were single housed in clean cages during the testing period with some beddings and nestlets from their home cages. Each mouse was represented as one data point. 4 (out of 113) mice in the post-injection test were excluded for bottle weighing problem/playing with the bottle/not trying water at all. 2 outliers were identified and excluded.

Chen R., Weitzner A.S., McKennon L.A, & Fonken L.K. (2021). Light at night during development in mice has modest effects on adulthood behavior and neuroimmune activation. Behavioural brain research, 405, 113171.

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