One ml of blood was lysed with 2 ml of ACK hypotonic solution (0.5M NH4Cl, 10 mM KHCO3, 200 μl 0.5M EDTA pH 8) after a 2-minute incubation at room temperature. Cells were washed twice in PBS (Sigma-Aldrich. St. Louis, USA), and the pellet was resuspended in 1 ml of RPMI (Invitrogen, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FCS. 10 6 cells were resuspended in 1 ml of medium, and used for cytospin preparation, while 2-3 x 10 6 cells were lysed in TRIzol (Invitrogen, Life Technologies, Carlsbad, CA, USA) and stored at -20°C. The rest of the blood samples were diluted 1:2 in sterile PBS (Sigma-Aldrich. St. Louis, USA), and layered over a cushion of 10 ml of Histopaque-1.077 (Sigma-Aldrich. St. Louis, USA). After centrifugation at 800 g for 45 minutes at room temperature, the mononuclear cells layer was collected, washed twice in sterile PBS (Sigma-Aldrich, St. Louis, USA), and centrifuged at 600 g for 10 minutes at room temperature. The pellet of mononuclear cells (PBMCs) was resuspended in 2 ml of RPMI (Invitrogen, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FCS. The PMNs layer was recovered and transferred into a sterile tube. Red blood cells were lysed using ACK hypotonic solution and washed again in sterile PBS (Sigma-Aldrich, St. Louis, USA). After centrifugation at
Sterile pbs
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, Italy, Canada
Sterile PBS is a laboratory product manufactured by Merck Group. It is a phosphate-buffered saline solution that is sterile and ready-to-use. The core function of Sterile PBS is to provide a balanced salt solution for various laboratory applications.
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Lab products found in correlation
Fine step stereotactic device, by Stoelting (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Trypan blue solution, by Merck Group (2 mentions)
Balb c mice, by Charles River Laboratories (2 mentions)
48 well suspension cell culture plates, by Sarstedt (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Comet assay kit, by Merck Group (1 mentions)
Annexin 5 kit, by Merck Group (1 mentions)
Bca kit, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Elisa kit, by Merck Group (1 mentions)
Albumin, by Merck Group (1 mentions)
Superdex 200 increase 10 300 gl, by Cytiva (1 mentions)
55 protocols using sterile pbs
1
Isolation and Purification of PBMCs and PMNs
2
CANDI Nanoparticle Therapy for Tumor Regression
3
Cytotoxicity Evaluation of Natural Compounds
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Albumin, chlorogenic acid (CGA), alcohol, acridine orange, ethidium bromide (EtBr), bovine serum Albumin (BSA), sterile coverslips, tissue culture plates, MTT, DMSO, propidium iodide, Annexin V kit, Comet assay kit, ELISA kits, antibiotics for cell culture (penicillin/streptomycin), sterile PBS, Ca2+, Mg2+ free PBS, RIPA buffer, trypsin/versene, BCA Kit, DCFH-DA kit, and other analytical grade chemicals were acquired from Merck, Darmstadt, Germany and Abcam chemical company, Waltham, USA. Bacterial cultures and MDA-MB-435s cell lines were purchased from NCCS, Pune, India.
4
Antibody-LP5 Conjugation Protocol
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50 μL of the antibody solution at 10.0 mg/mL in P5-conjugation buffer (50 mmol/L Tris, 1 mmol/L EDTA, 100 mmol/L NaCl, pH 8.3 at room temperature) were mixed with 1.66 μL of a 10 mmol/L TCEP [Tris(2-carboxyethyl)phosphine] solution (5 eq.) in P5-conjugation buffer. Directly afterwards, 0.83 μL of a 40 mmol/L solution of LP5 dissolved in DMSO (10 eq.) were added. The mixture was shaken at 350 rpm and 25°C for 16 hours. The reaction mixtures were purified by preparative size-exclusion chromatography (SEC) with a Superdex 200 Increase 10/300GL (Cytiva) and a flow of 0.8 mL/min eluting with sterile PBS (Merck). The antibody-containing fractions were pooled and concentrated by spin-filtration (Amicon Ultra 2mL MWCO: 30 kDa, Merck).
5
Cytotoxic Effects of Stimulators Assessed
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Cytotoxic effects of stimulators used in this study were evaluated using a tetrazolium yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which is reduced by living cells to yield soluble purple formazan crystals that can be detected colorimetrically. AGS cells and guinea pig fibroblasts (1 × 106 cells/well) were placed in 96-well plates in a volume of 100 μL and left to adhere overnight. Subsequently, all cells were washed with cRPMI and incubated for further 24 h with the described stimuli. Fresh MTT solution (5 mg/mL in sterile PBS; Sigma St. Louis, MI, United States) was added to each well and the plates were incubated for 2 h at 37 °C, 5% CO2. Formazan crystals were dissolved with acidic isopropanol (0.1 mol/L HCl in absolute isopropanol). Absorbance at 570 nm was estimated with a plate reader Victor2 (Wallac, Oy, Turku, Finland). All results were presented as the percentage means ± SD (standard deviation) relative to untreated cells of at least four independent experiments performed in triplicates. The effectiveness of MTT reduction was calculated based on the following formula: MTT reduction relative to untreated cells (%) = (absorbance of treated cells/absorbance of untreated cells × 100%) - 100%.
6
Disinfecting Specimen Preparation
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Specimens were sterilized by ethanol (eth, 70% in ddH2O) immersion. Briefly, discs were seeded onto a 12 multiwell plate and submerged with 1 ml/each of 70% eth for 2 h at room temperature; then, specimens were carefully washed 3 times with sterile PBS (1 ml/each from Sigma-Aldrich, Milan, Italy) and transported to a new multiwell plate. Plate was stored at room temperature protected from light with aluminum foil until experiments.
7
Anaerobic Bacterial Motility Assay
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Motility was assessed using the qualitative assay of Stabler et al.50 (link) with modification. As, Ans, Hs and Hns isolates were grown anaerobically on FAABL for 48 h. Working in the anaerobic chamber, triplicate colonies of each isolate were removed and resuspended in 50 µl sterile PBS (Sigma Aldrich, Irvine, UK) and a ten-fold serial dilution (undiluted to 10−4) was carried out. 10 ml of pre-reduced BHI containing 0.175% (w/v) agar was inoculated with each dilution, by carefully dropping 10 µl onto the surface, with minimum disturbance of the medium. Broths were incubated without shaking for 24 h under anaerobic conditions before being removed and photographed.
8
Downregulation of miR-218 In Vivo
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A locked Nucleic Acid (LNA) oligonucleotide with a sequence targeting miR-218 (Ant-miR-218) was used to downregulate the expression of miR-218 in vivo (Exiqon, Vedbaek, Denmark, Supplementary Table 1 ). A scrambled LNA oligonucleotide sequence was used as control (Ant-scrambled). Ant-miR-218 or Ant-scrambled were dissolved in sterile PBS (Sigma-Aldrich, Oakville, ON, Canada) at a final concentration of 0.3 mM, as indicated by25 (link), 26 . We confirmed the efficacy and specificity of Ant-miR-218 (Supplementary Figure 1 ) by quantitative PCR as in26 .
9
Downregulation of miR-218 In Vivo
10
Bioluminescent Quantification in Malaria Model
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Bioluminescent luciferase signal was quantified by imaging the whole animals using the in vivo IVIS 200 imaging system (Caliper Life Sciences, USA), as described elsewhere36 (link)64 (link)65 (link). Briefly, 44 hrs after the intravenous injection with 1000 transgenic P. berghei sporozoites the mice were anaesthetized in batches of three using isofluorane, their fronts shaved and D-luciferin (Synchem Laborgemeinschaft OHG, Germany) injected into the neck at a concentration of 100 mg/kg in sterile PBS (Sigma, US). Animals were imaged for 120 seconds at binning value of 8 and FVO of 12.8 cm, 8 minutes after the injection of D-luciferin. Mice were kept anaesthetized throughout the whole procedure. Quantification of bioluminescence signal was performed using Living Image 4.2 software (Caliper Life Sciences, USA). The region of interest (ROI) were set around the liver area of the mouse body and kept constant for all of the animals. The measurements were expressed as a total flux of photons per second of imaging time.
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