Tumor tissues were triturated and digested with trypsin–EDTA (11668-500, Invitrogen) to obtain single-cell suspension. Cells were cultured in ultralow attachment six-well plates (CLS3471-24EA, Corning) overnight. Appropriately 1 × 106 cells were taken used for flow cytometry and then incubated with 50 μL primary antibodies (anti-F4/80, 86007S, CST; anti-NOS2, 12-59520-82, Thermo Fisher; anti-CD163, 11-1631-82, Thermo Fisher) in an ice bath for 30 min in the dark. After incubation, cells were concentrated at 200 g for 5 min and removed supernatant. Finally, the precipitation was resuspended in PBS solution after washing and subjected to flow cytometry analysis within 1 h by FACSVerse™ (BD).
Cls3471 24ea
Manufactured by Corning
The CLS3471-24EA is a laboratory equipment product manufactured by Corning. It is designed for general laboratory use. The core function of this product is to provide a controlled environment for various laboratory experiments and processes. Detailed technical specifications and intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Anti adherence rinsing solution, by STEMCELL (2 mentions)
Aggrewell eb formation medium, by STEMCELL (2 mentions)
Aggrewell 400 24, by STEMCELL (2 mentions)
Tesr e6 media, by STEMCELL (2 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (2 mentions)
Aggrewell 400, by STEMCELL (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
4 protocols using cls3471 24ea
1
Characterizing Tumor-Associated Macrophages
2
Embryoid Body Formation using Aggrewell
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Embryoid bodies (EB) were formed using the Aggrewell-400 protocol according to the manufacturer’s protocol (STEMCELL Technologies). Briefly, the iPSCs were dissociated with Gentle Cell Dissociation Reagent (100–0485, STEMCELL Technologies) and seeded into the Aggrewell 400 24-well plate pre-coated with the anti-adherence rinsing solution (07010, STEMCELL Technologies) at a density of 1.2e6 cells/well in Aggrewell EB Formation Medium (05893, STEMCELL Technologies). After 24hr, half of the media was replaced with fresh Aggrewell EB Formation Medium. 48 hr after seeding, we harvested EBs and moved them to an ultra-low attachment 6-well plate (CLS3471-24EA, Corning) in TeSR™-E6 media (05946, STEMCELL Technologies). We maintained EBs in culture for 6 days, replacing media every other day.
3
Embryoid Body Formation using Aggrewell
4
Tumor Cell Isolation from Peritoneal Fluids
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Tumor cell clusters from patients with CRC (with a 50% mucinous contingent) and pseudomyxoma are harvested by collecting serous fluid after addition and reabsorption of 500 ml of saline solution immediately after laparotomy. Ascites from patients with esogastric, pancreatic, ovarian, and uterus cancer are collected at day hospital. The processing of peritoneal effusions is adapted from the protocol described in (13 (link)). Briefly, fresh specimens are centrifuged twice at 400g for 5 min, and tumor cells are isolated via a Ficoll-Paque PLUS (GE HealthCare, 17144002) centrifugation, following the manufacturer’s protocol. Cell clusters are then purified from single cells through several pulse centrifugations at 400g.
The nonseminomatous germ cell tumor specimen was isolated from biopsy. Tumoroids are kept in Matrigel for 6 weeks in colon organoid medium (41 (link)). They are then gently resuspended with TrypLE 1× and kept in suspension in an ultralow adhesion six-well plate (Corning, CLS3471-24EA) overnight before use in the microchannels.
The nonseminomatous germ cell tumor specimen was isolated from biopsy. Tumoroids are kept in Matrigel for 6 weeks in colon organoid medium (41 (link)). They are then gently resuspended with TrypLE 1× and kept in suspension in an ultralow adhesion six-well plate (Corning, CLS3471-24EA) overnight before use in the microchannels.
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