A total of 107L.g.− or L.g.+ metacyclic parasites were incubated with 1 mL of RPMI medium supplemented with 10% FBS and 10 μM of CFSE (CellTraceTM CFSE Cell Proliferation Kit, Invitrogen). The suspensions were incubated for 1 h at 37 °C and homogenized in every 15 min. Then, parasites were washed with 10 mL of fresh PBS1X (Gibco) and ressuspended with 5 mL of PBS1X, and left for 30 additional minutes at 37° to allow de-esterification of the dye. Finally, parasite suspensions were centrifuged (4000 rpm/10 min) and ressuspended in appropriate volumes for the infection. BMDMs were washed after 1 h of infection and left in culture up to 96 h. The data were acquired on a FACS ACCURI C6 flow cytometer (BD Biosciences) and analyzed with the FlowJo software (Tree Star).
Pbs 1x
Manufactured by Thermo Fisher Scientific
Sourced in United States, France, Italy
PBS 1X is a commonly used buffer solution that maintains a balanced salt concentration and pH level. It is a sterile, ready-to-use solution that is often employed in various laboratory applications, such as cell culture, immunoassays, and protein purification.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Pbs 1x, by Merck Group (4 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (4 mentions)
Varioskan lux, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (2 mentions)
96 well plate, by Corning (2 mentions)
Oil red o, by Thermo Fisher Scientific (2 mentions)
Oil red o, by Merck Group (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Dihydrorhodamine 123 dhr123, by Merck Group (2 mentions)
Kaluza flow cytometry software, by Beckman Coulter (2 mentions)
Crystal violet, by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Pore size cell strainers, by BD (2 mentions)
Pbs 1x, by Roche (2 mentions)
Pbs 1x, by GE Healthcare (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
72 protocols using pbs 1x
1
CFSE-based Parasite Proliferation Assay
2
Dose-Response of Essential Oils on Cells
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To evaluate the dose-response at given concentrations and the time-course of Eos, the cells where detached with Trypsin-EDTA 0.5% (Gibco, USA) and 5 × 103 cells/well were seeded and incubated in a 96-well cell plate with DMEM-F12 supplemented with 10% FBS for 24 h. The supernatant were removed and the cells were washed 1× with PBS 1X (Gibco, USA), and treated with EOs extracts of CA or LS at known concentrations (64, 32, 16, 8, 4, or 2 µg/mL) dissolved in culture medium (DMEM-F12 supplemented with 1% FBS). The vehicle was DMSO 1% dissolved in DMEM-F12, supplemented with 1% FBS as a control treatment (the EOs are dissolved in DMSO 1%). After 24 or 48 h the supernatant was discarded, the cells were washed with PBS 1X and incubated with 100 µl of crystal violet (CV) solution (0.2% w/v in ethanol 10%) (Gibco, USA) for 20 min, then the CV solution was removed and Na2HPO4 (0.1 M, pH 4.5, in ethanol 50:50 v/v) (SIGMA, USA), was added to elute the intra-cellular colorant. The absorbance of each sample was measured at 570 nm. The results are shown as percentage of color intensity and normalized to cells grown in DMSO 1% as control treatment.
3
Bovine Chondrocyte Viability Assay
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The viability of bovine articular chondrocytes was analysed using a Live & Dead viability/cytotoxicity kit (Thermo Fisher Scientific Inc., USA). Chondrocytes were plated at a density of 1 × 104 cells per well in flat black 96-well plates (Sarstedt, Germany). After 24 h of incubation under standard conditions (37 °C, 5% CO2), the cells were washed with PBS 1X (Life Technologies, Italy), and the ALD, HA + ALD mixture and HA-ALD solutions at six different dilutions (ALD final concentration: 100 μM, 50 μM, 25 μM, 12 μM, 6 μM, 3 μM) were added to the wells (three replicates were tested for each condition). Chondrocytes in DMEM/F-12 + 10% FBS were used as live controls, whereas chondrocytes treated with 70% methanol for 30 min were used as dead controls. The cells were incubated for 24 h, 3 days and 7 days, and then the medium was aspirated. The cells were washed with PBS 1X, and 100 μL of a 2 μM calcein-AM and 4 μM EthD-I solution in DMEM/F-12 w/o FBS was added. The plate was incubated for 20 min in the dark, and images were taken using an inverted fluorescence microscope (DMI8, Leica Microsystems, Germany). Samples were tested in three replicates. Live and dead cells were counted for each sample per condition, and finally, the mean number of viable cells per condition was normalized to the viability of untreated cells (control), which was set to 100%.
4
Senescence β-Galactosidase Staining
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Cells cultured on glass coverslips were tested for SA-β-galactosidase activity using the Senescence β-galactosidase Staining Kit (Cell Signaling, Danvers, MA) according to the manufacturer’s protocol. After 48 hours of treatment, cells were washed with phosphate buffered saline (PBS) 1x (Life Technologies, Carlsbad, CA), then fixed and stained with the provided reagents. The blue color is the reporter of senescence.
5
Recombinant Expression and Purification of PfTRiP
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DNA sequences encoding tRip (148–402) and tRip (181–402) either from P. berghei or P. falciparum were introduced into pCI‐neo vector for expression in mammalian cells. COS‐7 cells were cultured at 37°C with 5% CO2 in DMEM GlutaMAX growth medium (Life Technologies) containing 4.5 mg/L glucose, 50 U/ml penicillin, 50 μg/ml streptomycin and 10% fetal calf serum. They were transfected with pCI‐neo constructs and pCDNA3.1‐GFP as a control, using the jetOPTIMUS transfection reagent according to the manufacturer's instructions (Polyplus transfection). After 24 h incubation, cells were washed with PBS 1X (Life Technologies) and collected for further investigations. Cell lysis was achieved by three freezing and thawing cycles, in 5 mM Tris–HCl pH 8.0, in 0.1 M Na2CO3 pH 11.5 or in 5 mM Tris–HCl pH 8.0 containing 1% (v/v) Triton X‐100. Samples were incubated 30 min on ice before centrifugation (15 min at 15,000g). Pellets and soluble fractions were then separated on 10% SDS‐PAGE and transferred to PVDF membrane (Millipore) and analyzed by Western Blot with specific antibodies raised against PftRip (214–402). (Bour et al., 2016 (link)).
6
NBT Assay for Listeria Monocytogenes
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2x105 BMMC were cultured in 0.25 mL of RPMI-1640 without phenol red (Life Technologies, USA) and then, stimulated with L.m at different MOI for 120 minutes. In the last 15 minutes of incubation 25 μL of a solution of p-nitro blue tetrazolium (NBT) (Sigma-Aldrich, USA) at 1 mg/mL were added. Then, cells were washed with PBS 1X (Life Technologies, USA) and fixed with absolute methanol. The formazan precipitates were dissolved by adding 54 μL of potassium hydroxide (KOH) 2mM and 46 μL of Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA). The samples were read at a wavelength of 620 nm in a plate reader (Multiskan EX, Thermo Scientific, USA).
7
Peptide Vaccine Immunization in Mice and Rabbits
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Rabbits received 50 μg of the peptide W614A-3S coupled with CRM197 by the intramuscular route at Week 0 (W0), W2, W4, and W10. Mice received 10 μg of peptide coupled with keyhole limpet hemocyanin (KLH; Covalab) carrier protein or 11.7 μg of W614A-3S peptide coupled with CRM197 (Minka Therapeutics, Ile-de-France, France) by intramuscular injections (in both quadriceps of left and right thighs) at the same kinetic (or W18 for mouse CRM197 experiment). W614A-3S peptide (NH2-PWNASASNKSLDDIW-COOH) is a mutated peptide of HIV-1 gp41 (9 (link)). The antigen was administered with PBS1X (Life Technologies, Carlsbad, CA, USA), aluminum hydroxide (Alum; 100 μg for mouse and 250 μg for rabbit; Alhydrogel 2.0%; InvivoGen, San Diego, CA, USA), or squalene emulsion (SQE; 2.5% v/v for mouse and rabbit; Polymun, Klosterneuburg, Austria).
8
PCNA Immunohistochemistry in Tissue Sections
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For immunohistochemistry (IHC), sections were deparaffinized and hydrated. Serial slides were used for PCNA (Proliferating Cell Nuclear Antigen) staining. Tissue sections were treated with 0.1% triton X-100 (Sigma-Aldrich) in PBS 1x (Life Technologies) at room temperature (RT) for 10 min. Then endogenous peroxidases were inhibited by incubation with 3%H2O2 in methanol at RT for 10 min. Tissue sections were then placed in an antigen retrieval solution (0.01 M citrate buffer, pH = 6 (Zytomed, Berlin, Germany) for 15 min at 350 W) and quenched for endogenous peroxidases as described above. After saturation (GBI labs, Washington, USA), mouse anti-PCNA (M0879, DakoCytomation, Courtaboeuf, France) at 525 µg/ml was applied for 1 h at 37 °C. Sections were incubated with a kit anti-mouse HRP (GBI labs) for 30 min at RT. Staining was developed with HRP Green (Zytomed). Then, sections were counterstained with nuclear fast red (H3403, VectorLabs, Burlingame, CA, USA), dehydrated and mounted. Isotype control antibodies are used as negative controls.
9
Biocompatibility Evaluation of ALD and HA-ALD
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The biocompatibility of the tested compounds was evaluated by means of a quantitative analysis according to the ISO 10993–5:2012 International Standard. For cell viability assays, Saos-2 osteoblasts and primary bovine chondrocytes were plated at a density of 1 × 104 cells per well in 96-well plates (Corning, USA). After 24 h of incubation under standard conditions, the cells were washed with PBS 1X (Life Technologies, Italy), and solutions of ALD and HA-ALD were added to reach a final conc. of 3, 6, 12, 25, 50 and 100 μM (four replicates were tested for each condition). The cells were incubated for 24 h, 3 days and 7 days, and the medium was then aspirated. The cells were washed again with 1X PBS, and 100 μL of complete medium containing 10% Alamar Blue (Life Technologies, Italy) was added. The cells were subsequently incubated for 4 h under standard culture conditions, and finally, the fluorescence was measured using a microplate reader (Nanoquant Infinite M200 Pro, Tecan Group Ltd, Switzerland) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Samples were tested in four replicates. Values of IC50, the concentration of sample required to inhibit cell growth by 50% in comparison with the growth of a cell control, were analysed using Origin 8.5.1 software (Origin Lab Corporation, USA) with a non-linear sigmoidal fit.
10
Obtaining H. pylori-conditioned media
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To obtain conditioned media from gastric epithelial cells infected with H. pylori strains, GC cell lines were cultured in 12-well plates (Corning, Corning, NY, USA) in their respective medium at 1.5 × 105 cells per mL. H. pylori colonies were harvested and resuspended in PBS 1x (Life Technologies, Grand Island, NY, USA) to quantify the bacterial density at 550 nm. Bacterial suspensions were adjusted to infect the GC cell lines at a multiplicity of infection of 100 in their respective serum-free medium for 6 h at 5% of CO2 and 37 °C. H. pylori was then eliminated by treating with gentamicin (300 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 1 h; the GC cell lines were washed twice with PBS 1x and maintained for 18 h in their respective serum-free medium to complete 24 h at 5% of CO2 and 37 °C. Then, conditioned media were collected, centrifuged at 1500 rpm/5 min to eliminate floating cells, and used immediately to evaluate the chemoattraction of the different human B lymphocyte cell lines and for the IL-8 quantification.
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