Both MSCs LYS and MSCs CM were tested in vitro for their anti-proliferative activity on MSTO-211H, NCI-H2452 and NCI-H2052 cells in 96 multi-well plates (Sarstedt, Nümbrecht, Germany), as previously described [23 (link),24 (link)]. Briefly, 1:2 serial dilutions MSCs LYS and MSCs CM were performed in 100 µL of culture medium/well, and then, 103 tumor cells were added to each well. Cell growth was evaluated after 7 days of culture by measuring the optical density at 550 nm in an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazoliumbromide) assay [25 (link)]. Cell death was assessed by Hoechst 33342 and propidium iodide dual staining and by using the Apoptosis/Necrosis Detection Kit (Abcam, Cambridge, UK). Caspase-3 activity was measured by the Caspase-3 Assay Kit (Abcam) following the supplier’s protocols. The distribution of the cells in the cell cycle (determined by PI staining and flow cytometry analysis) was determined as described elsewhere [26 (link)].
96 multiwell plates
Manufactured by Sarstedt
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96 multiwell plates are laboratory equipment used for high-throughput screening and analysis. They provide a standardized format with 96 individual wells to hold and process small volumes of samples or reagents simultaneously.
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Lab products found in correlation
Caspase 3 assay kit, by Abcam (1 mentions)
Apoptosis necrosis detection kit, by Abcam (1 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
Spectrafluor plus, by Tecan (1 mentions)
Mtt assay, by Promega (1 mentions)
Universal microplate reader elx800nb, by Agilent Technologies (1 mentions)
8 protocols using 96 multiwell plates
1
Evaluating the Anti-Proliferative Effects of MSCs on Mesothelioma Cells
2
HSV-1 Infection of SH-SY5Y Neuroblastoma Cells
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The SH-SY5Y neuroblastoma cell line (n. 94030304, Merck, Darmstadt, Germany), derived from a four-year-old, female children was selected as a neuronal model for HSV-1 infection, since the susceptibility of these cells to HSV-1 infection was previously observed [15 (link),16 (link)].
The cells were maintained in 44.5% MEM and 44.5% Ham’s F12, supplemented with 10% fetal bovine serum, non-essential amino acid (10 mM for each amino acid), 2 mM glutamine and 100 U/mL penicillin/streptomycin (all the reagents were from Euroclone, Pero, Milan, Italy). A total of 104 cells were seeded each day prior to infection in 96-multiwell plates (Sarstedt, Munich, Germany) in 100 μL of final volume. After 24 h they were infected with different quantities of viral DNA copies of HSV-1, i.e., 5 × 106, 5 × 105, 5 × 104, 5 × 103, and incubated for 24 h or for 48 h at 37 °C and 5% CO2.
The cells were maintained in 44.5% MEM and 44.5% Ham’s F12, supplemented with 10% fetal bovine serum, non-essential amino acid (10 mM for each amino acid), 2 mM glutamine and 100 U/mL penicillin/streptomycin (all the reagents were from Euroclone, Pero, Milan, Italy). A total of 104 cells were seeded each day prior to infection in 96-multiwell plates (Sarstedt, Munich, Germany) in 100 μL of final volume. After 24 h they were infected with different quantities of viral DNA copies of HSV-1, i.e., 5 × 106, 5 × 105, 5 × 104, 5 × 103, and incubated for 24 h or for 48 h at 37 °C and 5% CO2.
3
Assessing Mycelium Early Development and Biomass
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Mycelium early development (post-germination hyphal growth) was assessed by inoculating 5 × 103 of CR10 conidia in 96 multiwell plates (Sarstedt, Newton, NC, USA), in a final volume of 200 μL/well of YES liquid medium amended with test compounds at increasing concentrations (from 25 to 50 µM), and analysing changes in optical density after 46 h of static growth at 28 °C. DMSO (0.25, 0.5 and 1% v/v respectively) was used as control. The optical density was recorded at 620 nm for each well with a microplate reader (TECAN SpectraFluor Plus microplate reader, Männedorf, Switzerland) without shaking. Samples were inoculated in quadruplicate. Values were then converted to percentage inhibition with respect to the relevant control (DMSO-treated cultures), and expressed as means ± S.D.
Mycelium biomass production was assessed after six days of incubation: mycelia from single wells were recovered, slightly dried on paper towels, and weighed. Values were then converted to percentage inhibition with respect to the relevant control (DMSO-treated cultures), and expressed as means ± S.D.
Mycelium biomass production was assessed after six days of incubation: mycelia from single wells were recovered, slightly dried on paper towels, and weighed. Values were then converted to percentage inhibition with respect to the relevant control (DMSO-treated cultures), and expressed as means ± S.D.
4
Evaluating Anti-Tumor Effects of MSCs-PTX/CM
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Conditioned media from MSCs-PTX (MSCs-PTX/CM) were prepared seeding MSCs-PTX in 48 multiwell plates (24,000 cells/well, 350 µL/well); the plates were maintained at 37 °C, 5% CO2, and Conditioned Media (CM) were collected after 24, 48, 72, 96, 120 h after seeding. The effect of pure PTX and MSCs-PTX/CM against tumor cell proliferation was studied in 96 multiwell plates (Sarstedt, Numbrecht, Germany) by using as target pancreatic adenocarcinoma cells (CFPAC-1) and a mesothelioma cell line (NCI H2052). Briefly, 103 tumor cells were seeded in each well in 100 µL of culture medium/well and after 24 h MSCs-PTX/CM was added to each well. A serial dilutions curve of pure drug as well as MSCs-PTX/LYS were plated as controls. After 7 days of culture at 37 °C and 5% CO2 cell growth was evaluated by MTT assay (Promega, Medison, WI, USA), as previously described [29 (link)].
5
Evaluating Paclitaxel's Anticancer Potential
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The effect of pure paclitaxel and CM from PTX-treated ASCs (CM/PTX) against tumour cell proliferation has been studied in 96-multiwell plates (Sarstedt, Germany) by using as target pancreatic adenocarcinoma cells (CFPAC-1) [17 (link)]. Briefly, 1 : 2 serial dilutions of pure drug or ASC-CM were prepared in 100 μl of culture medium/well and then to each well were added 1000 tumour cells. After 7 days of culture at 37°C and 5% CO2, cell growth was evaluated by MTT assay (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium) as previously described [5 (link), 18 (link)]. The inhibitory concentrations (IC50 and IC90) were determined according to the Reed and Muench formula [19 (link)]. The antitumor activity of PTX CM was compared to that of pure PTX and expressed as PTX equivalent concentration: PEC (ng/ml) = IC50 PTX × 100/V50 (μl/well); IC50 PTX = the concentration of pure PTX producing 50% of inhibition; V50 is the volume PTX-MSCs-CM able to inhibit cell proliferation by 50%. The PEC referred to a single primed ASC was calculated as ratio between the total amount of PEC (PEC (ng/ml) × CM volume (ml)) and the number of cells seeded: PE release (pg/cell) = PEC (ng tot)∗1000/number of cells seeded. Uptake release experiments were performed in triplicate.
6
Amaranth Extracts' Impact on RAW 264.7 Cell Viability
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The viability of RAW 264.7 cells incubated with variable concentrations of amaranth extracts was assayed with MTT test. Cells (5 × 105 cells/well) were seeded onto the 96 multi-well plates (Sarstedt, Numbrecht, Germany) and kept for 24 h as adherent cultures at 37 °C. Then, different dry methanol amaranth extracts dissolved in DMSO were added to the appropriate cell wells, and incubation was performed for 24 h. Six concentrations of amaranth extracts: 1 mg/mL; 500 μg/mL; 100 μg/mL; 10 μg/mL; 1 μg/mL; 0.1 μg/mL of medium were tested. The cells cultured in medium were used as positive control (100 % of growth), whereas the cells incubated with addition of 20 mM/L hydrogen peroxide to medium provided the negative control (0 % of growth). The test was conducted as described previously [32 (link)]. The absorbance was measured at 550 nm (the reference wavelength of 690 nm) using Universal Microplate Reader ELX800NB (Bio-Tek Instruments INC, Winooski, VT, USA). The following formula was applied to report the results: (average OD value over three measurements for each experimental group/average OD value of control group) × 100 %. Each experiment was repeated in triplets.
7
Antiproliferative Effects of CisPt and Pt-8AQ
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The effect of CisPt and Pt-8AQ against cell proliferation has been studied in 96 multiwell plates (Sarstedt, Nümbrecht, Germany). Briefly, 1:2 serial dilutions of pure drug (from 0.52 to 64 µM CisPt and from 0.27 ± 35.52 µM Pt-8AQ) were prepared in 100 µL of culture medium per well, and then to each well, we added 2000 tumor cells. After 7 days of culture (anti-proliferative assay) at 37 °C and 5% CO2, cell growth was evaluated by MTT assay (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium), as previously described. [18 (link),19 (link)]
The in vitro stability of CisPt and Pt-8AQ was analyzed, maintaining the drugs at 37 °C for 24 h; then, the drugs were tested on U87-MG cells in anti-proliferation assay and compared to fresh drugs. Cytotoxicity assay (24 h at 37 °C, 5% CO2) was performed at increasing concentrations of 2.5, 5, 10, and 20 μM. The inhibitory concentration (IC50) was determined by applying the Reed and Muench formula [20 (link)] using Excel (Microsoft, Inc, Albuquerque, NM, USA).
The in vitro stability of CisPt and Pt-8AQ was analyzed, maintaining the drugs at 37 °C for 24 h; then, the drugs were tested on U87-MG cells in anti-proliferation assay and compared to fresh drugs. Cytotoxicity assay (24 h at 37 °C, 5% CO2) was performed at increasing concentrations of 2.5, 5, 10, and 20 μM. The inhibitory concentration (IC50) was determined by applying the Reed and Muench formula [20 (link)] using Excel (Microsoft, Inc, Albuquerque, NM, USA).
8
Quantifying PTX Release from MFAT and DMFAT
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To evaluate the amount of PTX released by MFAT-PTX and DMFAT-PTX specimens, we performed a biological dosage of the drug by evaluating the PTX equivalent concentration (p-EC) [17] . Briefly, the effect of both free PTX and CM, derived from MFAT-PTX and DMFAT-PTX specimens cultured for 24h (37°C, 5% CO 2 ) in IMDM complete medium, on tumor cell proliferation was studied in 96 multiwell plates (Sarstedt, Germany) by using as target CFPAC-1 cells. Serial dilutions (1:2) of drugs or CM from treated and control untreated fat specimens were prepared and added to 1,000/100 µl tumor cells. After 7 days of culture at 37°C, 5% CO 2 cell viability was evaluated by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium) assay. The inhibitory concentrations (IC) 50 and 90 were determined according to the Reed and Muench formula [34] , and also by using the linear regression analysis on the dose-response kinetics of the inhibition. The anti-tumor activity of CM from MFA-PTX and DMFAT-PTX were compared to the one of pure PTX and expressed as PTX equivalent concentration (p-EC) according to the following algorithm p-EC (ng/ml) = IC50 PTX x 100/V50 (ul/well) where IC50 PTX is the concentration of pure PTX producing 50% growth inhibition and V50 the respective volume of CM that produce the same inhibition.
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