Human auricular chondrocytes from the rapid or slow cell groups were suspended in 1% atelocollagen at a concentration of 1 × 107 cells/mL. The cell-collagen mixture (20 μL) was placed at the bottom of a 15 mL conical tube (BD Falcon, USA) and incubated for 2 h at 37 °C and 5% CO2. Differentiation medium (2 mL) (DMEM/F12 supplemented with insulin-like growth factor-1 [IGF-1]; 1 μg/mL; Astellas Pharma Inc. Tokyo, Japan) was added after gelation, and the cells were cultured for 3 weeks in an incubator at 37 °C and 5% CO2.
15 ml conical tube
Manufactured by BD
Sourced in United States
The 15 ml conical tube is a laboratory equipment item used for various applications in scientific research and testing. It is a cylindrical container with a tapered bottom, designed to hold a liquid volume of up to 15 milliliters.
Automatically generated - may contain errors
Lab products found in correlation
Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (4 mentions)
Transfer buffer, by Thermo Fisher Scientific (4 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (4 mentions)
Nupage loading buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf pre cut blotting membranes, by Thermo Fisher Scientific (4 mentions)
Tube revolver, by Thermo Fisher Scientific (3 mentions)
Moflo astrios eq, by Beckman Coulter (3 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions)
7 aminoactinomycin d, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by BD (2 mentions)
70 μm nylon mesh, by BD (2 mentions)
Hygromycin b, by Thermo Fisher Scientific (2 mentions)
T 175 flask, by BD (2 mentions)
Nylon mesh filter, by BD (1 mentions)
Tc20 automated cell counter, by Bio-Rad (1 mentions)
Crystallized dii, by Thermo Fisher Scientific (1 mentions)
Tungsten beads, by Bio-Rad (1 mentions)
Prestoblue, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Varioskan lux, by Thermo Fisher Scientific (1 mentions)
18 protocols using 15 ml conical tube
1
Chondrocyte Culture in Atelocollagen
2
Lung tissue dissociation and single-cell sorting
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Lung tissue was transported in Hank’s balanced salt solution (HBSS, Life Technologies) on ice immediately after surgery. Half of the tissue was embedded, and the rest was cut into 1-mm3 pieces and digested with collagenase I (2 mg/mL) and IV; (1 mg/mL) in a 15 mL conical tube (BD Falcon) at 37°C for 30 min on a tube revolver (Thermo) with frequent agitation. All samples were then filtered through 70 μm and 40 μm nylon mesh filters (BD Biosciences) and centrifuged at 4°C at 400 x g for 5 min. The cell pellet was suspended in red blood cell lysis buffer, centrifuged and resuspended in PBS with 0.04% FBS. Following dissociation, single-cell suspensions were stained with 7-aminoactinomycin D (7-AAD) in a dark room for 15 min before being analyzed by flow cytometry for live-cell sorting with a MoFloAstrios EQ (Beckman Coulter). Cell suspensions were added to the Master Mix to achieve a final number of 8000 cells per reaction for scRNA-seq.
3
Cell Counting Protocol for NSC Cultures
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We used 4 NSC culture plates per experiment, which were prepared with the same number of cells and maintained under identical culture conditions. For cell counting, one of these plates was randomly selected at 24 hrs, 48 hrs and 72 hrs of culture, transferred to a 15 ml conical tube (BD Falcon), centrifuged and then treated with TrypLE for 5 minutes after removing the supernatant. Dissociated cells were stained with trypan blue and counted using a TC20 automated cell counter (Bio-Rad Laboratories, CA, USA).
4
HEK293-T Cell Transfection and Protein Analysis
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Example 8
The transformation of HEK293-T cells with the prepared O1P1-3C-SGLuc constructs was performed as previously described herein. The incubation media was removed and assayed for luciferase activity as previously described herein. Cells were removed from surface by repeated pipetting of media then collected in a 15 ml conical tube (Falcon) and centrifuged at 500 rpm for 5 min to pellet cells. Cells were then re-suspended in 200 μl of MPER mammalian protein extraction reagent (Invitrogen). Samples were mixed with 4× NuPAGE loading buffer (Invitrogen) per manufacturer's instructions and heated at 95° C. for 10 min. Samples were then briefly centrifuged and loaded onto NuPAGE Novex 4-12% Bis-Tris protein gels (Invitrogen) and run in 1×MES (2-(N-morpholino)ethanesulfonic acid) buffer at 200 V for 35 min. The harvested, transformed cells were transferred to 0.2 m pore size PVDF pre-cut blotting membranes (Invitrogen) with 1×transfer buffer (Invitrogen) per manufacturer's instructions.
5
Protein Extraction and SDS-PAGE Analysis
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Example 8
The transformation of HEK293-T cells with the prepared O1P1-3C-SGLuc constructs was performed as previously described herein. The incubation media was removed and assayed for luciferase activity as previously described herein. Cells were removed from surface by repeated pipetting of media then collected in a 15 ml conical tube (Falcon) and centrifuged at 500 rpm for 5 min to pellet cells. Cells were then re-suspended in 200 μl of MPER mammalian protein extraction reagent (Invitrogen). Samples were mixed with 4× NuPAGE loading buffer (Invitrogen) per manufacturer's instructions and heated at 95° C. for 10 min. Samples were then briefly centrifuged and loaded onto NuPAGE Novex 4-12% Bis-Tris protein gels (Invitrogen) and run in 1×MES (2-(N-morpholino)ethanesulfonic acid) buffer at 200 V for 35 min. The harvested, transformed cells were transferred to 0.2 μm pore size PVDF pre-cut blotting membranes (Invitrogen) with 1× transfer buffer (Invitrogen) per manufacturer's instructions.
6
Transformed HEK293-T Cell Protein Analysis
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Example 8
The transformation of HEK293-T cells with the prepared O1P1-3C-SGLuc constructs was performed as previously described herein. The incubation media was removed and assayed for luciferase activity as previously described herein. Cells were removed from surface by repeated pipetting of media then collected in a 15 ml conical tube (Falcon) and centrifuged at 500 rpm for 5 min to pellet cells. Cells were then re-suspended in 200 μl of MPER mammalian protein extraction reagent (Invitrogen). Samples were mixed with 4×NuPAGE loading buffer (Invitrogen) per manufacturer's instructions and heated at 95° C. for 10 min. Samples were then briefly centrifuged and loaded onto NuPAGE Novex 4-12% Bis-Tris protein gels (Invitrogen) and run in 1×MES (2-(N-morpholino)ethanesulfonic acid) buffer at 200 V for 35 min. The harvested, transformed cells were transferred to 0.2 μm pore size PVDF pre-cut blotting membranes (Invitrogen) with 1× transfer buffer (Invitrogen) per manufacturer's instructions.
7
Transformation and Analysis of HEK293-T Cells
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Example 8
The transformation of HEK293-T cells with the prepared O1P1-3C-SGLuc constructs was performed as previously described herein. The incubation media was removed and assayed for luciferase activity as previously described herein. Cells were removed from surface by repeated pipetting of media then collected in a 15 ml conical tube (Falcon) and centrifuged at 500 rpm for 5 min to pellet cells. Cells were then re-suspended in 200 μl of MPER mammalian protein extraction reagent (Invitrogen). Samples were mixed with 4× NuPAGE loading buffer (Invitrogen) per manufacturer's instructions and heated at 95° C. for 10 min. Samples were then briefly centrifuged and loaded onto NuPAGE Novex 4-12% Bis-Tris protein gels (Invitrogen) and run in 1×MES (2-(N-morpholino)ethanesulfonic acid) buffer at 200 V for 35 min. The harvested, transformed cells were transferred to 0.2 μm pore size PVDF pre-cut blotting membranes (Invitrogen) with 1× transfer buffer (Invitrogen) per manufacturer's instructions.
8
DiOlistic Labeling of Neuronal Structures
9
Single-cell isolation from resected tumors
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Resected tumors were transported in Hank’s Balanced Salt Solution (HBSS, Life Technologies) on ice immediately after surgery. The tumor sample was subsequently divided into two pieces, and a small fragment was stored in liquid nitrogen for tissue staining. The remainder of the tumor was minced with scalpels into tiny cubes <0.5 mm3 and transferred into a 15 mL conical tube (BD Falcon) containing 8 mL pre-warmed HBSS, 1 mg/mL collagenase I and 0.5 mg/mL collagenase IV. Tumor pieces were digested on a Tube Revolver (Thermo) for 30 min at 37 °C. This suspension was then filtered using a 70 μm nylon mesh (BD Biosciences) and residual cell clumps were discarded, then the cell pellet was resuspended in red blood cell lysis buffer. Following a 5 min incubation at room temperature, samples were centrifuged to discard the supernatant and re-suspend the cell pellet in PBS with 0.04% FBS. Cell sorting was performed with a MoFloAstrios EQ (Beckman Coulter). Live cells were used for single-cell experiments after the dead cells were eliminated based on exclusion of 7-aminoactinomycin D (Life Technologies).
10
Optimizing hiPSC Culture Conditions
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The hiPSC line 19c3 (p20–p60), derived from a healthy male, was used for medium variable optimization. Prior to assay, cells were grown to 75% confluence after 4 days of culture as above. Cells were dissociated with TrypLE (Gibco, 12604013) for 3 min at 37°C and resuspended in DMEM/F12, transferred to a 15-mL conical tube (Falcon), and centrifuged at 200 × g for 3 min (Sorvall ST40). The pellet was resuspended in DMEM/F12 and diluted to 1 × 105 cells mL−1, and 10,000 cells were plated per well in Matrigel (1:800)-coated 12-well plates (Greiner) in the medium to be tested along with 2 μM thiazovivin for the first 24 h. Media were changed daily and cells were grown for 6 days. This lower than normal seeding density was used to allow the discovery of factors detectable only under more extreme conditions and therefore provide data on the robustness of the formulation. Cell growth was then assessed using PrestoBlue (Invitrogen, A13262), by adding 100 μL of PrestoBlue to the 1 mL of existing medium in each well and incubating for 2 h at 37°C. Fluorescence (560 nm excitation, 590 nm emission) was then measured using a Varioskan LUX (Thermo Scientific) plate reader with “top read” function.
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