Ascites obtained from the peritoneal tap of patients with ovarian cancer was provided by Sri Sankara Cancer Hospital with due ethical clearance. Patient spheroids were cultured in tissue culture-treated polystyrene substrata/polyHEMA coated dish using DMEM (AL007A; HiMedia)—supplemented with 10–20% FBS (10270; Gibco) and antibiotics or with defined medium. Spheroids were then collected from the cultures by centrifugation at 0.1–0.2g for 5 min.
Al007a
Manufactured by HiMedia
The AL007A is a laboratory equipment designed for general laboratory use. It has a basic function of providing a stable and controlled environment for various scientific experiments and procedures. The specific details of its core function and intended use are not available at this time.
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7 protocols using al007a
1
Ovarian Cancer Patient-Derived Spheroids
2
Ovarian Cancer Cell Line Cultivation
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The human ovarian cancer cell lines: OVCAR3, OVCAR4, and SKOV3 were a kind gift from Professor Rajan R. Dighe, Indian Institute of Science. OVCAR3 and OVCAR4 were maintained in DMEM (AL007A; HiMedia) supplemented with 10–20% FBS (10270; Gibco) and antibiotics in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The SKOV3 cell line was maintained in McCoy’s 5A medium (AL275A; HiMedia) supplemented with 10% FBS and antibiotics. The ovarian cancer cell line G1M2 (patient-derived xenograft line) was a kind gift from Professor Sharmila A. Bapat, the National Centre for Cell Science (NCCS), India. G1M2 cell line was maintained in Roswell Park Memorial Institute medium (AL162A; HiMedia) supplemented with 10% FBS and antibiotics.
3
Evaluating Insulinoma Cell Viability
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A 200 μl of INS-1-rat insulinoma cell line cell (Sigma-Aldrich) was suspended in RPMI medium (#AT150, Himedia) with 5% fetal bovine serum ((#RM10432, Himedia). This was seeded onto a 96-well plate at a cell density of 8,000 cells/well. The cells were allowed to grow overnight followed by washing with Dulbecco’s phosphate buffer saline (PBS). Serum-free media was added followed by incubation for 3 h. The extract was added at different concentrations and PBS-treated cells were used as a negative control. The plates were incubated for 48 h at 37 °C in a 5% CO2 atmosphere, with or without glucose (20 mM). Following this, the spent media was taken out and Eagle’s minimal essential medium (100 μl) (#AL007A, Himedia) containing 10% fetal calf serum and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (# 4060, Himedia) at a concentration of 0.5 mg/ml was added. The mixture was again incubated at 37 °C for 30 min without exposure to light. A solubilizing agent (dimethyl sulfoxide-100 μl) was added after removing the MTT reagent. The experiments were conducted in triplicates and optical density was determined at 540 nm.
4
HEK293T Cell Lysis and Protein Extraction
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HEK293T cells were grown in DMEM (AL007A, Himedia) supplemented with 10% FBS (RM1112, Himedia) and antibiotic-antimycotic agent (15,240,062, Gibco). Cells were collected and washed with PBS (TL1006, Himedia). Washed cell pellet was lysed in RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, 1% NP40 (IGEPAL), 0.1% Na-deoxycholate and 0.1% SDS) containing Halt protease and phosphatase inhibitor cocktail (PI78441, Invitrogen).
5
HEK293T Cell Lysis Protocol
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Cell culture and cell lysate preparation HEK293T cells were grown in DMEM (AL007A, Himedia) supplemented with 10% FBS (RM1112, Himedia) and antibiotic-antimycotic agent (15240062, Gibco). Cells were collected and washed with PBS (TL1006, Himedia). Washed cell pellet was lysed in RIPA buffer (150 mM NaCl, 5 mM EDTA , 50 mM Tris, 1% NP40 (IGEPAL), 0.1% Na-deoxycholate and 0.1% SDS) containing Halt protease and phosphatase inhibitor cocktail (PI78441, Invitrogen).
6
HEK293T Cell Lysis Protocol
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Cell culture and cell lysate preparation HEK293T cells were grown in DMEM (AL007A, Himedia) supplemented with 10% FBS (RM1112, Himedia) and antibiotic-antimycotic agent (15240062, Gibco). Cells were collected and washed with PBS (TL1006, Himedia). Washed cell pellet was lysed in RIPA buffer (150 mM NaCl, 5 mM EDTA , 50 mM Tris, 1% NP40 (IGEPAL), 0.1% Na-deoxycholate and 0.1% SDS) containing Halt protease and phosphatase inhibitor cocktail (PI78441, Invitrogen).
7
Ovarian Cancer Cell Line Cultivation
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Ovarian cancer cell lines used in this study are OVCAR3 and G1M2 (patient derived xenograft line). These cell lines were maintained in Dulbecco's Modified Eagle's Medium -high glucose (AL007A; HiMedia) and Roswell Park Memorial Institute medium (AL162A; HiMedia) supplemented with 10-20 % FBS and recommended antibiotics in a humidified atmosphere of 95 % air and 5 % CO2 at 37°C.
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