All reagents and cell culture material were of analytical grade or of the highest grade available and were acquired from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibiotic-antimycotic (AB-AM) was acquired from Grisp (Porto, Portugal), Trypsin/EDTA 2.5%, 3,3′-Dihexyloxacarbocyanine Iodide (DiOC6) from Gibco/Invitrogen Co. (Carlsbad, CA, USA), Pierce Lactate Dehydrogenase (LDH) cytotoxicity assay kit from Thermo Fisher (Waltham, MA, USA), and Dibutylphthalate Polystyrene Xylene (DPX) from VWR-Prolabo (Radnor, PA, USA). Culture flasks were from Sarstedt (Nümbrecht, Germany) and all the other plastic materials used in cell culture techniques were either from Falcon (Tewksbury, MA, USA) or Nerbe plus (Winsen, Germany).
Culture flasks
Manufactured by Sarstedt
Sourced in Germany
Culture flasks are laboratory vessels used for the cultivation and maintenance of cell or microbial cultures. They provide a controlled environment for the growth and study of various biological specimens.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta solution, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by LGC (2 mentions)
Pierce lactate dehydrogenase ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Non essential amino acids (neaa), by Harvard Bioscience (1 mentions)
Amphotericin b, by Harvard Bioscience (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Recombinant human il 1β, by InvivoGen (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Pam2csk4, by InvivoGen (1 mentions)
Hcv29, by Merck Group (1 mentions)
Tccsup, by American Type Culture Collection (1 mentions)
Hcv29, by American Type Culture Collection (1 mentions)
Tccsup, by LGC (1 mentions)
6 protocols using culture flasks
1
Cytotoxicity Assay Protocol for Cell Lines
2
Epothilone D Compound Preparation
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Chemicals, cell culture media, supplements, culture flasks, plates, dishes and other plastic material were obtained from Sarstedt (Nümbrecht, Germany), Sigma-Aldrich (Deisenhofen, Germany) and Thermo Fisher Scientific (Waltham, USA), unless otherwise stated. Epothilone D (EpoD) was a kind gift from Amos Smith 3rd (University of Pennsylvania) and was prepared as previously described [27 (link), 28 (link)]. The spectroscopic properties of the compound were identical to those reported in the literature. Compound purity was >95% as determined by LC-MS and NMR analyses.
3
Isolation of Rabbit ACL Fibroblasts
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Lapine ACL samples were explanted from dead New Zealand White (NZW) rabbits (female donors, age around 11 weeks), sacrificed in other approved experimental animal projects (including LaGeSo Berlin 396/09, 21 January 2010). Three native ACLs were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin for immunohistology. For cell isolation, surrounding connective tissue of the ACLs, including the synovial membranes, was removed. The pure ligament tissue was cut into 1–2 mm2 slices and incubated in culture flasks (Sarstedt, Nümbrecht, Germany) with growth medium (Ham’s F-12/Dulbecco’s Modified Eagle’s (DMEM) Medium 1:1) containing 10% fetal calf serum (FCS), 10,000 IU/mL penicillin / 10,000 µg/mL streptomycin, 2.5 µg/mL amphotericin B, non-essential amino acids (all from Biochrom AG, Berlin, Germany), and 25 µg/mL ascorbic acid (Sigma-Aldrich, Munich, Germany) at 37 °C and 5% CO2. After 1−2 weeks, the ACL fibroblasts started to migrate from the tissue slices. Subsequently, fibroblasts were harvested using 0.05% trypsin/0.02% Ethylenediaminetetraacetic acid (EDTA) (Biochrom AG, Berlin, Germany) and sub-cultured.
4
Induction of M-MDSC-like Cells from Monocytes
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To induce M‐MDSC‐like cells, the isolated primary monocytes from each donor were divided into 2 fractions and seeded (1.5 × 106/cm2) in culture flasks (Sarstedt, Nümbrecht, Germany), either stimulated with LPS (LPS‐B5 (E. coli serotype 055: K59(B5)H‐), 10 ng/ml Invivogen, San Diego, CA) for 20 h (M‐MDSC‐like cells) or left untreated (normal monocytes). Other stimuli used, such as recombinant human IL‐1β (E. coli expressed human IL‐1β with HSA), Pam2CSK4 (synthetic diacylated lipoprotein), and Pam3CSK4 (synthetic triacylated lipopeptide), were also purchased from Invivogen. Following a medium change, both fractions of cells were then allowed to rest for 40 h. Following the resting period, the cells were reseeded and were challenged with 10 ng/ml LPS. A schematic presentation of the timeline can be seen in Figure 1 .
5
Bladder Cancer Cell Culture Protocol
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Two human bladder cancer cell lines were
used: nonmalignant cells of the ureter (HCV29, derived from the Institute
of Experimental Therapy, PAN, Wroclaw, Poland) and transitional cell
carcinoma (TCCSUP, ATCC, LGC Standards). HCV29 cells were cultured
in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum
(FBS, Sigma). TCCSUP cells were cultured in Eagle’s minimum
essential medium (EMEM, LGC Standards) supplemented with 10% FBS.
These different media represent the physiological conditions specific
to each cell line. Cells were grown in culture flasks (Sarstedt) in
an incubator (Nuaire) at 37 °C in 95% air and 5% CO2 and relative humidity above 98%. Cells were passaged when their
confluence reached 80–90%. HCV29 and TCCSUP cells were detached
from the surface using 0.05% and 0.25% trypsin–EDTA solution
(Sigma) for 4 min, respectively. After a few passages, cells were
seeded on a Petri dish (passages 6–8) for AFM measurements
and on glass coverslips for fluorescence imaging.
used: nonmalignant cells of the ureter (HCV29, derived from the Institute
of Experimental Therapy, PAN, Wroclaw, Poland) and transitional cell
carcinoma (TCCSUP, ATCC, LGC Standards). HCV29 cells were cultured
in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum
(FBS, Sigma). TCCSUP cells were cultured in Eagle’s minimum
essential medium (EMEM, LGC Standards) supplemented with 10% FBS.
These different media represent the physiological conditions specific
to each cell line. Cells were grown in culture flasks (Sarstedt) in
an incubator (Nuaire) at 37 °C in 95% air and 5% CO2 and relative humidity above 98%. Cells were passaged when their
confluence reached 80–90%. HCV29 and TCCSUP cells were detached
from the surface using 0.05% and 0.25% trypsin–EDTA solution
(Sigma) for 4 min, respectively. After a few passages, cells were
seeded on a Petri dish (passages 6–8) for AFM measurements
and on glass coverslips for fluorescence imaging.
6
Bladder Cancer Cell Lines for Research
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Three human bladder cancer cell lines were used in this study: HCV29 -non-malignant transitional epithelial cancer cell of the ureter (obtained from the Institute of Experimental Therapy, PAN, Wroclaw, Poland), T24 -transitional cell carcinoma (ATCC, LGC Standards) and HT1376 -grade III urinary bladder cell carcinoma (ATCC, LGC Standards). HCV29 and T24 were cultured in RPMI-1640 medium (Sigma) supplemented with 10% of fetal bovine serum (FBS, Sigma). HT1376 were grown in Eagle's minimum essential medium (EMEM, LGC Standards) with 10% FBS. All cell lines were grown in culture flasks (Sarstedt) in an incubator (Nuaire) at 37° C in 95% air and 5% CO2 atmosphere. The relative humidity was kept above 98%. The cell passaging was carried out when they reached 80-90% of the confluency level. For HCV29 and T24 cells, 0.05% and for HT1376 cells, 0.25% of trypsin-EDTA solution (Sigma) was applied. After a few passages (< 10), cells were ready to be seeded on a Petri dish or glass coverslips for fluorescence imaging and AFM measurements. STR profile of HCV29 cell line: amelogenin: X,Y; TH01: 6,8; D13S317: 8,11; D16S539: 12,13; D5S818: 12; D7S820:
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