Skeletal muscle stem cells were isolated from hind limb muscles of 6- to 8-week-old mice (kindly provided by P. Muñoz Cánoves of the Pompeu Fabra University, Barcelona) as described in [75 (link)]. After mechanical and enzymatic dissociation with 1 % Pronase protease (Calbiochem), the filtered digest was centrifuged through an isotonic Percoll (Amersham) gradient (60 % overlaid with 20 %) and cells were collected from the gradient interface, resuspended in growth medium (GM: Ham’s F-10 plus 20 % FBS, 5 ng/ml bFGF, 100 U/ml penicillin and 100 μg/ml streptomycin) and grown on tissue culture dishes coated with 0.05 mg/ml collagen I from rat tail (Becton and Dickinson) to amplify the myoblast population. To induce myotube formation, confluent proliferating primary myoblasts were grown on matrigel coated culture dishes (Basement Membrane Matrix, Becton and Dickinson) and switched to differentiation medium the next day (DM: DMEM plus 2 % horse serum, 100 U/ml penicillin and100 μg/ml streptomycin (Life Technologies)) for 4 days. Myofibres directly isolated from EDL muscles were kindly provided by S. Gutarra. Two biological replicates of each experiment were performed and analysed. Mouse protocols were approved by the Animal Care and Use Committee of the PRBB, and the Ethical Committee for Animal Experimentation of the Government of Catalonia.
Basement membrane matrix
Manufactured by BD
Sourced in United States, United Kingdom
Basement Membrane Matrix is a solution of extracellular matrix proteins designed for use in cell culture applications. It provides a complex mixture of basement membrane components to support the growth and differentiation of various cell types.
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Lab products found in correlation
Ix73 microscope, by Olympus (7 mentions)
Zoletil 100, by Virbac (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Recombinant human vegf a, by R&D Systems (2 mentions)
Mouse anti phospho erk1 2 y204, by Santa Cruz Biotechnology (2 mentions)
Anti phospho fak y397, by Abcam (2 mentions)
Rabbit anti ho 1, by Enzo Life Sciences (2 mentions)
Insulin syringes 30g 3 10 cc, by BD (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Penicillin, by BD (1 mentions)
Streptomycin, by BD (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Imiquimod r837, by InvivoGen (1 mentions)
Interleukin 6 (il 6), by Beyotime (1 mentions)
Tnf α, by Beyotime (1 mentions)
Pe anti mouse cd11c, by BioLegend (1 mentions)
Fluorescein isothiocyanate (fitc), by MedChemExpress (1 mentions)
Il 12, by Beyotime (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
55 protocols using basement membrane matrix
1
Isolation and Differentiation of Murine Skeletal Muscle Stem Cells
2
Immune Response Evaluation Protocol
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Imiquimod (R837) and Indooyanine gree (ICG) were purchased from Invivogen and BBI Life Science Corporation, respectively. Ovalbumin (OVA), calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI) were purchased from Sigma-Aldrich Inc. FITC was purchased from MedChemExpress LLC and Cy5.5 was purchased from APExBIO Technology LLC. Anti-mouse CD274 (B7-H1, PD-L1), FITC-anti-mouse CD86, PE-anti-mouse CD11c, TITC-anti-mouse CD8 and PerCP-anti-mouse CD3ε for flow cytometry were purchased from Biolegend Inc. Basement Membrane Matrix was purchased from Becton, Dickinson and Company. TNF-α, IL-6, IL-12 and IFN-γ ELSA kit were purchased from Beyotime Biotech Inc.
3
Transwell Invasion Assay for Cancer Cells
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Transwell inserts (membrane pore size, 8 μm; diameter, 6.4 mm; cat. no. 353097, FALCON®, Corning Inc., United States) were placed in 24-well plates. The cells were seeded in serum-free medium in upper chambers (150,000 cells per chamber for U87 and U87-TxR; 70,000 cells per chamber for C6 and RC6) covered with a Matrigel layer (500 ng/ml; cat. no. 356234, Basement Membrane Matrix, BD Biosciences, United States) and subsequently treated with 2 μM 5 and 8 μM 6 . The lower chambers were filled with appropriate medium supplemented with 10% fetal bovine serum as a chemoattractant. A negative control without 10% fetal bovine serum was also included in each experiment, as a measurement of spontaneous cell invasion. After 24 h, cells that invaded through the Matrigel and membrane were fixed in 4% paraformaldehyde. Cells remaining on the upper surface of the membrane were carefully removed with a cotton swab. Invading cells from the lower surface of the membranes were stained with Hoechst 33342. Membranes were carefully removed from inserts and placed on microscopic glass slides. Stained cells were counted under a Zeiss Axio Vert inverted fluorescent microscope at 10× magnification. Stained cells were counted in ImageJ software (1.48, Microsoft, Redmond, WA, United States).
4
Evaluating Antitumor Immune Memory with E7 Nanofibers
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TC-1 cells (1×105) mixed with Basement membrane matrix (BD Biosciences, San Jose, CA, USA) were injected subcutaneously (s.c.) into the right flank of the C57BL/6 mice to establish the HPV-associated grafted tumor model. A preventive immunization strategy was employed as described in Figure 2A . Mice were first immunized s.c. with 12.5 nmol of E744-62-Q11 or Q11 nanofibers or unassembled E744-62-Q11 peptides three times at an interval of 2 weeks (n = 5 mice per group) and then challenged with TC-1 cells 2 weeks after the last immunization. To assess the effective antitumor immune memory induced by nanofibers, the mice were rechallenged with TC-1 6 weeks after the first TC-1 cell inoculation (Figure 2A ). In the therapeutic studies, the mice were first challenged with TC-1 cells. When the tumor diameter reached 2–3 mm or 5–6 mm, three immunizations were performed at an interval of 7 days (n =6 per group) (Figure 3A and 4A ). The tumor growth was measured every 3–4 days using a micrometre caliper. Tumor volumes were calculated using the following formula: volume (mm3) = 0.5 × (width [mm])2 × length [mm]. Mice were euthanized when the largest tumor diameter reached 20 mm. At the end of each experiment, 4 mice were randomly selected from each group, and splenocytes were isolated for analyses on lymphocyte and cytokine responses.
5
Quantifying Lung Metastasis in NOD-SCID Mice
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NOD-SCID mice (6–8 weeks, male) were purchased from the National Laboratory Animal Center (Tainan, Taiwan). The mice were anesthetized by intraperitoneal injection of Zoletil® 100 (Virbac, France). Stable shRNA-expressing cells (2 × 105 CL1-5-Luc/GFP-shLacZ or shBMP2 cells) were resuspended in a solution containing 10 µL PBS and 10 µL Basement Membrane Matrix (BD, USA) and directly injected into the right lung via BD Insulin Syringes (30G 3/10 cc, BD, USA). The body weights of xenograft model mice were measured three times per week until sacrifice. All procedures complied with the animal care standards set forth by the guidelines of the NCKU Laboratory Animal Center (Tainan, Taiwan). The whole mouse lung tissue with shLacZ and shBMP2 groups was scanned by TissueFAXS. The total area of metastasis colonies in left lung was quantified using the Image J software(1.53 K).
6
Cell Invasion and Migration Assay
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To evaluate cell invasion and migration, cells were cultured in the presence or absence of basement membrane matrix (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The transfected cells were cultured in the upper chamber (3×104 cells/100 μL) with serum-free medium, while cells in the lower chambers were cultured in 20% fetal bovine serum (8-mm pore; Corning, Life Science, Lowell, MA, USA). The cells were incubated for 24 h at 37 °C with 5% CO2 and then stained with 1% crystal violet (20 min; room temperature); cell numbers in five random regions were counted. The extent of cell invasion and cell migration was determined as mean ± standard deviation of the number of cells per field.
7
Xenograft Tumor Induction in Mice
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Control and mutant cybrid cells (1.5*106) were resuspended in Basement Membrane Matrix (Matrigel™ BD Biosciences) and injected subcutaneously into the lateral flank of adult NMRI nu/nu mice (28–30 g). Tumor growth was monitored until reaching a volume of 1.2 cm3.
8
Xenograft Tumor Formation from iPSCs
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Gln-iPSCs were harvested by accutase treatment, collected into tubes, and centrifuged. The same volume of Basement Membrane Matrix (354234, BD Biosciences) was added to the cell suspension. The cells (>5.0×106) were subcutaneously inoculated into immunodeficient mice (BALB/cAJcl-nu/nu, CREA, Tokyo, Japan). After 6 to 12 weeks, the resulting tumors were dissected and fixed with formalin. Paraffin-embedded tissue was sliced and stained with hematoxylin and eosin (HE). The operation protocols were accepted by the Laboratory Animal Care and the Use Committee of the National Research Institute for Child and Health Development, Tokyo. For comparison, we used iPSCs of healthy and diseased donors. iPSCs such as PAE-iPSCs, UtE-iPSCs, AM-iPSCs, and 201B7 have been generated from placental, endometrial, amniotic, and fibroblastic cells [8 (link), 9 (link), 31 (link)–34 (link)].
9
Tube Formation Assay in HDLECs
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Tube-formation assays were performed as described previously.50 (link) Briefly, conditioned HDLECs (1.5 × 104) were diluted with 100 μL of serum-free RPMI 1640 in each well of a 96-well culture plate precoated with 50 μL of basement membrane matrix (BD Biosciences, Franklin Lakes, NJ, USA). The plate was then incubated at 37°C for 6 h and tube formation visualized under an inverted microscope (CX41; Olympus, Tokyo, Japan). The length of tube structures was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
10
Regulation of Cell Invasion by miR-23a
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Aspc-1 cells were transfected with either miR-23a mimics alone or miR-23a mimics with the ESRP1 over-expression vectors for 48 h. Panc-1 cells were transfected with either miR-23a inhibitors alone or miR-23a inhibitors with ESRP1 siRNA for 48 h. For the cell invasion assay, transwell chambers were coated with 50 μg reconstituted basement membrane matrix (BD Biosciences, MD, USA). Then, 1 × 104 cells in 100 μl serum-free medium were seeded into the upper chambers. Next, 600 μl of DMEM containing 5% FBS was added to the lower chamber. After 48 h of incubation, cells on the lower surface of the membrane were stained with crystal violet. The cells were counted in five random fields under a light microscope.
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