Forma series 2 water jacket co2 incubator

Primary human dermal fibroblasts were isolated from skin donated, with informed consent, by a healthy adult volunteer in accordance with the protocols of the Affiliated Hospital of Guilin Medical University. The fibroblasts were seeded at a density of approximately 2.2 × 10⁶ cells in 10 mL of complete culture media (CCM, comprising DMEM with 10% FBS and 1% P/S) in a 100 mm culture dish (Eppendorf, Hamburg, Germany) and cultured till confluence at 37 °C under 5% CO₂ (Forma Series II Water Jacket CO₂ Incubator, Thermo Scientific, Waltham, MA, USA) before they were used for further experiments.
The choice of primary human dermal fibroblasts for this study was grounded in their well-documented responsiveness to fibroblast growth factors and their critical role in wound-healing processes. There is currently no in vitro TMP model. Human dermal fibroblasts offer a reproducible model to assess the cytoproliferative and chemotactic effects of FGF-2, providing a foundational understanding crucial for the future development and application of FGF-2 formulations in tissue repair and regeneration. By recognizing the importance of cellular characterization in primary cultures, this approach allows us to establish a baseline for FGF-2′s therapeutic potential, acknowledging that detailed fibroblast characterization is essential for tailoring future drug applications.

B hermsii DAH 2E7 was cloned by limiting dilution in liquid mBSK-c medium from the low-passaged non-clonal isolate DAH [18] , [27] (link), [28] , [76] . Although much of the studies regarding antigenic variation in B. hermsii have been done with isolate HS1, the DAH isolate is nearly identical to HS1, as shown by multi-locus sequencing [27] (link) and plasmid sequences containing the silent vmp genes [16] (link), [18] . The vmp gene in the expression site of B. hermsii DAH 2E7 was amplified and sequenced with primers pro and tel (Table S1) as described [16] (link), [21] (link), [77] and was determined to be vlp7. Between vlp7 and the telomere is a vsp26 pseudogene, which is silent in B. hermsii HS1 [16] (link), [21] (link), [77] . Transformants of low-passaged DAH 2E7 were selected with kanamycin at 100 μg/ml or with gentamicin at 20 μg/ml in mBSK-c containing 12% rabbit serum (Pel-Freez, Rogers, AR) [28] , [78] (link), incubated at 35°C and 5% CO₂ and 3% O₂ in a Forma Series II Water Jacket CO₂ incubator (Thermo Fisher Scientific, Inc., Waltham, MA) with the caps loosely attached on the culture tubes. Clones were isolated by limiting dilution in liquid medium as described [28] . Escherichia coli TOP10 (Invitrogen, Carlsbad, CA) was used for the generation of constructs and grown in Luria broth or agar plates with kanamycin (50 μg/ml) or gentamicin (5 μg/ml).

HeLa, 293T and U373MG cells were cultured in DMEM supplemented with 10% Fetal Calf serum (FBS). BHK-U3GFP indicator FAG (Fluorescence Activated GFP) cells were cultured in DMEM supplemented with 5% FBS and 500 µg/mL G418 (Gibco). Leptomycin B (LMB) (Sigma) was added to culture medium to a final concentration of 10 nM for 4 h. Hypoxic conditions (2% O₂, 5%CO₂ and 93% N₂) were induced by a continuous flow of nitrogen using a Forma Series II Water Jacket CO2 incubator (model: 3131; Thermo Scientific).

The hypoxic conditions were created using a Forma Series II Water Jacket CO₂ incubator (model: 3131; Thermo Scientific), which allows precise oxygen and temperature regulation. The hypoxic conditions were generated at 37 °C in 5% CO₂ and the designated oxygen content, balanced with N₂. All of the media used in the hypoxia experiments were preincubated overnight in the chambers with the designated oxygen content.

HEK293 and SH-SY5Y cells (ATCC) were cultured in phenol red-free Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 2 mM L-Glutamine (Invitrogen), heat-inactivated 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Cell cultures were maintained in an incubator with 5% CO₂ (Forma Series II Water Jacket CO₂ Incubator, Thermo Scientific) at 37 °C. The inter-protomeric (oligomer) and intra-protomeric (oligomer and conformation) αSN FRET biosensors were generated by transiently transfecting HEK293 cells using Lipofectamine 3000 (Invitrogen) with GFP-αSN and αSN-RFP (1:8 DNA plasmid concentration ratio) or GFP-αSN-RFP plasmid, respectively. The effectiveness of HEK293 cells transfected with FRET constructs as an HTS platform has been demonstrated in our previous work^{46 (link)–52 ,66} .