Allegra x 12r centrifuge

Transfections of HEK293T cells were conducted by combining 2.5 μg of MSCV-IRES-GFP or MSCV-RUNX1-ETO9a-IRES-GFP retroviral expression vector, 2.5 μg of MSCV-IRES-tdTomato or MSCV-Rassf2-IRES-tdTomato, along with packaging components. For transduction, primary E16.5–E18.5 murine fetal liver cells were resuspended in supplemented retroviral supernatant at densities of 2–3 × 10⁶ cells/ml and centrifuged (2000 × g) in six-well plates at 32 °C for 3 h (Allegra X-12R centrifuge, Beckman Coulter), followed by overnight culture at 37 °C. Two consecutive retroviral transductions were performed in this manner on subsequent days. Transduction efficiencies were measured (GFP+ and tdTomato+ frequencies) by flow cytometry. Transduced cell populations were flow sorted and then pooled with untransduced “helper” fetal liver cells at a ratio of 1:6. For the primary leukemia model, 150,000 total cells per mouse were then transplanted into lethally irradiated (9.5 Gy) recipients and leukemia was monitored. Recipients were randomly allocated to experimental groups with equal distribution based on sex. Recipient mice found to develop GFP-negative/non-myeloid malignancies were excluded from analysis. Investigators were not blinded during group allocation or assessment of experimental outcomes.

All the chemicals and solvents (Sigma-Aldrich, Milan, Italy) were of analytical reagent grade and were used without any further purification. For the centrifugation of in-house cultured organisms, we used an Allegra X-12R centrifuge (Beckman Coulter, Milan, Italy).
The solid phase extraction was carried out using both prepacked and non-polystyrene–divinylbenzene columns (CHROMABOND^® HR-X, Macherey-Nagel, Düren, Germany). Automated fractionations were carried out on the GX-271 ASPEC Gilson apparatus equipped with TRILUTION^® LH Software (version No. 3.0, Gilson, Middleton, WI, USA). Silica gel chromatography was performed using precoated Merck F254 plates.
¹H NMR spectra were recorded on a Bruker DRX 600 spectrometer equipped with an inverse TCI CryoProbe or 400 equipped with a CryoProbe Prodigy. Extracts were dissolved in 700 µL CDCl₃/CD₃OD 1:1 (v/v) and transferred to the 5 mm NMR tube. Chemical shift was referred to CHD₂OD signal at δ 3.34.

Cell apoptosis was evaluated by flow cytometry using an Annexin V‐FITC Apoptosis Detection Kit (KeyGen Biotech Co. Roche, Nanjing, China). Briefly, cells were seeded into 24‐well plates at 1 × 10⁵ cells per well and cultured for 48 hours. Then, the cells were detached by trypsinization, washed twice in PBS (400 g, 5 minutes; Allegra X‐12R centrifuge; Beckman Coulter, USA) and resuspended in 500 μL binding buffer. A volume of 5 μL Annexin V‐FITC and 5 μL propidium iodide was added and mixed gently, and the cells were stained in the dark for 10 minutes at room temperature. The cells were analysed immediately by flow cytometry (BD FACSCalibur, BD Bioscience, San Diego, CA, USA) and analysed using Flowjo software (FlowJo, Ashland, OR, USA). The experiment was repeated three times.

T22 and BOL-12 were maintained on potato dextrose agar (BD-Difco, Detroit, USA) at 25°C. To isolate conidiospore suspensions, one ml of sterile water was added to two-week-old Trichoderma cultures on potato dextrose agar and collected spores were filtered through a sterile piece of cotton wool. The spores were washed twice with sterile H₂O (Milli-Q, Merck Millipore, Burlington, MA, USA) and pelleted at 3700g for 5 min at 4 °C in an Allegra X-12R centrifuge (Beckman, Brea, CA, USA). Spores were resuspended in sterile H₂O and kept at 4°C until experiments.
Germination of T22 and BOL-12 spores for C. quinoa treatment was performed as described by Yedidia, Benhamou (Yedidia et al., 1999 (link)) using 15 ml tubes shaken at 200 rpm for 18 h. The germinated spore suspension was washed twice by centrifugation as described above and finally resuspended in sterile H₂O. The final spore concentration was adjusted to be 1 germinated spore/μl and verified by colony forming unit (CFU) counts in potato dextrose agar Petri dishes.

G. africana was collected during the month of May 2015 from the Western Cape Province, South Africa. The plant was identified by Dr. Chris N. Cupido, the co-author of this paper, and a specimen was deposited in Kirstenbosch National Botanical Garden (Cape Town, South Africa) under accession number 1468255/NBG. The fresh aerial parts of G. africana were dried in the shade. To obtain the aqueous extract, 50.0 mL of boiled distilled water were added to 5.0 g of the dried plant powder. Afterwards, the plant decoction was centrifuged for 2 h at 3750 rpm using an Allegra^® X-12R centrifuge (Beckman Coulter, Cape Town, South Africa). The supernatant was then filtered through 0.45 μm filters and freeze-dried using FreeZone 2.5 L freeze-dryer (Labconco, Kansas City, MO, USA).

Cell apoptosis was evaluated by flow cytometry using an Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech Co. Roche, Nanjing, China). Briefly, cells were seeded into 24-well plates at 1 × 10⁵ cells per well and cultured for 48 h. Then the cells were detached by trypsinization, washed twice in PBS (2000 r.p.m., 5 min; Allegra X-12R centrifuge; Beckman Coulter, USA) and resuspended in 500 µl binding buffer. A volume of 5 µl Annexin V-FITC and 5 µl propidium iodide was added and mixed gently, and the cells were stained in the dark for 10 min at room temperature. The cells were analysed immediately by flow cytometry (BD FACSCalibur, BD Bioscience, San Diego, CA) and analysed using Flowjo software (FlowJo, Ashland, OR). The experiment was repeated three times.