6×103 semi-stable transfected cells were plated on 6-well dishes in appropriate medium supplemented with 3% agarose (Type VII, Sigma A4018) over a warm layer of pre-solidified medium containing 6% agarose (Type VII, Sigma A4018). Five days after plating the cells a new 1 cm layer of media was plated over the growing cells. HeLa cells containing 17-AGG had a final concentration of 8.8 ug/dL 17-AAG agar mixture. The ability of cells to grow on soft agar was evaluated after 14 days. Colonies were analyzed under the light microscope.
Type 7
Manufactured by Merck Group
Sourced in United States
Type VII is a laboratory equipment product from Merck Group. It is designed for a core function of sample preparation and processing. The detailed specifications and intended use of this product are not available in this limited context.
Automatically generated - may contain errors
Lab products found in correlation
9 protocols using type 7
1
Soft Agar Assay for Cell Growth
2
Soft Agarose Culture of T. equiperdum
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The T. equiperdum IVM-t1 strain, which was isolated in Mongolia in 2015, was used in the present study (Suganuma et al., 2016 (link)). Soft agarose culture-adapted T. equiperdum IVM-t1 was maintained by continuous sub-culturing once a week at 37 °C in 5% CO2 using soft agarose media (HMI-9 (Hirumi and Hirumi, 1991 (link)) with 0.8% low gelling agarose (wt/vol) [Type VII, Sigma-Aldrich Japan, Tokyo, Japan]).
3
Fixation and Embedding of HCT-116 Cells
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Culture of HCT-116 cells were centrifuged at 0.355 x g for 3 min and the supernatant was removed, afterwards the pellet was fixed in a 2% (w/v) formaldehyde and 0.2% (v/v) glutaraldehyde in 0.1 M PBS (pH 7.4, Sigma-Aldrich) for 90 min at room temperature. Then the cells were washed several times in PBS, after the last washing the pellet in PBS was warmed to 37°C and the same volume of 2% low gelling temperature agarose (type VII, Sigma-Aldrich) was quickly added to the cell suspension and placed in a refrigerator for 30 min. The hardened agarose blocks were cut under PBS into small pieces and dehydrated in graded ethanol series (50%, 70%, 90% and 96%; 30 min each) and infiltrated in 2:1 (v:v) and 1:2 (v:v) ethanol/LR White mixture, 30 min each on ice. Then the samples were infiltrated in pure LR White (Polysciences Inc., Warrington, PA, USA), after 2 h the resin was exchanged and allowed to stand overnight at 4°C. The resin was exchanged again on the next day and after 2 h the samples were placed into gelatin capsules and polymerized for 48 h at 50°C. Afterwards, thin sections with white interference color (70-100 nm) were cut using ultramicrotome OmU 3 (Reichert, Vienna, Austria) equipped with a diamond knife (45°; Diatom AG, Biel, Switzerland). The sections were mounted on Formvar coated 200 mesh nickel grids (Polysciences Inc.) and immuno-labelled.
4
Soft Agar Colony Formation Assay
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The 5-Fu-treated and parent MC3 cells were seeded in 24-well plates. Low melting-point agarose (0.3 ml, 0.6%; Type VII, Sigma-Aldrich, St. Louis, MO, USA) was poured into each well and 0.3 ml (0.35%) agarose containing 100 cells was subsequently added to each well. The cells were incubated following the solidification of agarose at room temperature. The number of clones containing >50 cells was counted under a microscope after ten days and the cloning efficiency was calculated using the following formula: Colony formation rate (%) = no. of clones/no. of cells incubated × 100.
5
Single-Cell Comet Assay for DNA Damage
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Single-cell gel electrophoretic comet assay was performed according to the previous study.61 (link) In brief, cells with or without 4-OHE2 treatment were recovered in a normal culture medium. Then cells were collected and rinsed twice with ice-cold PBS. In all, 2 × 104/mL cells were combined with 1% low-gelling-temperature agarose (Sigma; Type VII, cat.no. A-4018) at 40°C at the ratio of 1:3 (v/v) and immediately pipetted onto slides. For cellular lysis, the slides were immersed in the alkaline lysis solution (1.2 M NaCl, 100 mM Na2EDTA, 0.1% sodium lauryl sarcosinate, 0.26 M NaOH in pH > 13) overnight at 37°C in dark, followed by washing in the rinse buffer (0.03 M NaOH, 2 mM Na2EDTA) for 30 min with two repeats. Then, the slides were subjected to electrophoresis at 20 V (0.6 V/cm) for 25 min and stained in 2.5 μg/mL propidium iodide for 20 min. All images were taken with a fluorescence microscope (Olympus IX73) and analyzed by Comet Assay IV software.
6
Soft Agar Colony Formation Assay
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Cells (2.5 × 103) were suspended in BEBM, containing 0.35% low-melting agarose (Type VII, Sigma-Aldrich) and seeded onto 0.5% low-melting agarose in six-well tissue culture plates. Colonies were scored after 3 weeks in 5 randomly selected fields/well, at 4X magnification. The experiments were repeated at least three times.
7
Soft Agar Clonogenic Assay
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Cells (2.5 × 103) were suspended in RPMI medium containing 0.35% low-melting agarose (Type VII, Sigma-Aldrich) and seeded onto 0.5% low-melting agarose in six-well tissue culture plates. Colonies were scored after 3 weeks in 5 randomly selected fields/well, at 4X magnification. Photographs were also taken at higher magnification (40X) to measure clone size. Clones were classified according to their diameter in > 200 μm or <200 μm.
8
Gut Tissue Preparation for Microscopy
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The digestive tract was dissected and the mesentery was completely removed. The dissected guts were either used as such (tensile testing), or embedded in collagen gels (migration assays) or low-melting point agarose gels (Type VII, Sigma-Aldrich) at 37 °C. For AFM and SHG analyses, the guts were embedded in agarose and a double-blade cutter (Multirex, France) was used to cut 1 mm-thick transverse slices from different parts of the gut. Slices were immobilized by “gluing” them onto the base of a Petri dish with liquid agarose. The samples were hydrated with PBS, kept at 4 °C, and imaged within 5 h by AFM or SHG microscopy.
9
Osteochondral Defect Repair with MSCs
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The study was approved by Administrative Panel on Laboratory Animal Care (APLAC) of Stanford University and all the methods were carried out in accordance with the approved guidelines. In five athymic female Harlan rats, osteochondral defects were created in the distal femur of both knee joints under inhalation anesthesia. A medial patellar skin incision was made, the patella was dislocated laterally and a circular osteochondral defect (diameter: 2 mm, depth: 1.5 mm) was created in the distal femoral trochlear groove using a micro-drill (Saeyang, Daegu, Korea). In these defects, either 7.5 × 105 ferumoxytol labeled viable hMSCs (right femur, n = 5), or ferumoxytol labeled Mitomycin-pretreated hMSCs (left femur, n = 5) were implanted, using an agarose scaffold (5 μl, Type VII, Sigma Aldrich, St Louis, MO, USA). We have previously shown, that Mitomycin-pretreated hMSC undergo apoptosis in vivo within 24 h after transplantation21 (link)66 (link). After allowing the agarose scaffold to solidify for 1–2 minutes, the patella was repositioned and skin incision was closed by Dermalon 6-0 monofilament sutures.
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