Fibroblasts were isolated from a fresh foreskin, as described previously [28 (link)]. The isolated cells were cultured in DMEM (Capricorn scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS) (Capricorn scientific, Ebsdorfergrund, Germany) until passage three. Then at 80% confluency in a 75 cm2 cell culture flask (SPL life sciences, Pocheon, Korea), cells were washed twice with phosphate-buffered saline (PBS), and serum starvation conditions were started for 16 h in serum-free, glucose-containing DMEM (Capricorn scientific, Ebsdorfergrund, Germany) without antibiotics. Cell culture supernatants were collected, and stored at −20 °C for further analysis. The collected conditioned supernatant has been named as 16h-starved fibroblast culture supernatant (16h-SFS). The current protocol has been approved by the local ethical committee at Babol University of Medical Sciences (reference number: IR. MUBABOL.HRI.REC.1398.333).
75 cm2 cell culture flask
Manufactured by SPL Life Sciences
Sourced in United States
The 75 cm2 cell culture flask is a standard laboratory equipment used for the in vitro cultivation of cells. It provides a surface area of 75 square centimeters for cell attachment and growth.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Capricorn (1 mentions)
Dulbecco modified eagle medium (dmem), by Capricorn (1 mentions)
Roswell park memorial institute (rpmi), by Biowest (1 mentions)
Penicillin streptomycin, by PAN Biotech (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
C2c12 cell line, by Korean Cell Line Bank (1 mentions)
Dulbecco modified eagle medium (dmem), by PAN Biotech (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hb 8065, by American Type Culture Collection (1 mentions)
Nunclon delta surface, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Low glucose dmem, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline pbs, by Biowest (1 mentions)
Neubauer improved, by Marienfeld (1 mentions)
4 protocols using 75 cm2 cell culture flask
1
Isolation and Starvation of Fibroblasts
2
Human PBMC Secretome Analysis
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In this study we used the identical protein data set which has been published previously [29 ]. This data set was prepared from mass spectrometry analysis of human PBMCs culture supernatant which is briefly described in the next section.
PBMCs of three healthy donors, gender: F, age: ±SD: 26 years ± 2.64, were isolated by ficoll density gradient centrifugation as reported previously [29 ]. 7 × 106 isolated PBMCs were cultured in a 75 cm2 cell culture flask (SPL life sciences, Korea), in serum-free, glucose-containing RPMI (Biowest, Nuaillé, France) 1640 for 96 h without antibiotics. Then the cells were collected and centrifuged at 450 g for 10 min and finally the supernatant were frozen at −20 °C until mass spectrometry analysis. This solution was named as96 h- starved P BMC culture s upernatant (96h-SPS).
The ethic committee of Babol University of Medical Sciences approved this protocol under reference number IR. MUBABOL.HRI.REC, 1398.221.
PBMCs of three healthy donors, gender: F, age: ±SD: 26 years ± 2.64, were isolated by ficoll density gradient centrifugation as reported previously [29 ]. 7 × 106 isolated PBMCs were cultured in a 75 cm2 cell culture flask (SPL life sciences, Korea), in serum-free, glucose-containing RPMI (Biowest, Nuaillé, France) 1640 for 96 h without antibiotics. Then the cells were collected and centrifuged at 450 g for 10 min and finally the supernatant were frozen at −20 °C until mass spectrometry analysis. This solution was named as
The ethic committee of Babol University of Medical Sciences approved this protocol under reference number IR. MUBABOL.HRI.REC, 1398.221.
3
Culturing C2C12 Myoblast Cell Line
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The C2C12 cell line was obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, PAN biotech), supplemented with 10% fetal bovine serum (JCBIO) and 1% penicillin–streptomycin (PAN biotech). The cells were incubated at 37 °C in a standard atmosphere (95% air and 5% CO2) during all experiments. The cell culture was maintained with 9 × 105 cells in 75-cm2 cell culture flasks (SPL Life Sciences), and sub-cultured every 48 h after seeding by replacing the culturing media every day. The cell was cultured for three passages after thawing to stabilize the growth conditions29 (link).
4
HepG2 Cells Interaction with Isolated Platelets
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HepG2 cells (HB-8065, ATCC) were cultured in 75 cm2 cell culture flasks (SPL Life Sciences, Gyeonggi, Korea); cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/low glucose (Gibco, Green Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (P/S; Gibco) at 37 °C and 5% CO2. For subculture, HepG2 cells were washed first with Dulbecco’s phosphate buffered saline (PBS; Biowest, Nuaillé, France), and cell detachment was performed using 0.5% trypsin–EDTA (Invitrogen, Waltham, MA, USA), followed by resuspension in cell culture medium.
For the assays, 1 × 106 HepG2 cells were seeded into 6-well dishes (Nunclon Delta Surface; Thermo Scientific, Waltham, MA, USA). HepG2 cells were then counted using a Neubauer-improved (0.0025 mm2, depth 0.1 mm; Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) hemocytometer. After overnight incubation at 37 °C and 5% CO2, cells were washed using PBS and resuspended in calcium-free DMEM supplemented with 2% FBS and 1% P/S, followed by incubation with 1 × 107 isolated platelets for 6 h. Supernatants from each well were then collected in a 2 mL microtube, and the cells were detached using cell scrapers. All of the cells and supernatants were stored at − 20 °C until analysis.
For the assays, 1 × 106 HepG2 cells were seeded into 6-well dishes (Nunclon Delta Surface; Thermo Scientific, Waltham, MA, USA). HepG2 cells were then counted using a Neubauer-improved (0.0025 mm2, depth 0.1 mm; Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) hemocytometer. After overnight incubation at 37 °C and 5% CO2, cells were washed using PBS and resuspended in calcium-free DMEM supplemented with 2% FBS and 1% P/S, followed by incubation with 1 × 107 isolated platelets for 6 h. Supernatants from each well were then collected in a 2 mL microtube, and the cells were detached using cell scrapers. All of the cells and supernatants were stored at − 20 °C until analysis.
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