The largest database of trusted experimental protocols

695 protocols using kh2po4

1

Cultivation and Preparation of Dictyostelium Strains

Check if the same lab product or an alternative is used in the 5 most similar protocols

D. discoideum axenic strains used in the study were AX3 (Dictybase ID: DBS0235545), DH1 (Dictybase ID: DBS0302388),
phg2 (Dictybase ID: DBS0302388),
pdsA (Dictybase ID: DBS0237030), and
carA (Dictybase ID: DBS0236438). All the strains were cultured in autoclaved HL5 medium (per L, 5 g proteose peptone, 5 g thiotone E peptone, 5 g yeast extract, from USBIO, 10 g glucose, 0.35 g Na
2HPO
4*7H
2O, 0.35 g KH
2PO
4 from Sigma-Aldrich, pH=6.7) at 22°C if not mentioned otherwise. In experiments on nutritional effect we used: FM minimal medium (Formedium), NS (per L, 15.2 g peptone, 7.6 g yeast extract, from USBIO, 5mg Na
2HPO
4, 5mg KH
2PO
4, from Sigma-Aldrich, pH=6.7) and NS with 85mM glucose (Sigma-Aldrich) added after autoclaving
15 (link). The bacterial species used as the nutritional source in our study was
Klebsiella aerogenes. Heat killed bacterial cultures were prepared by centrifuging 50mL of overnight LB cultures at 4°C, 5000 g for 10min and diluting the pellet in 1mL KK2 buffer (per L, 22 g KH
2PO
4, 7.0 g K
2HPO
4, Sigma-Aldrich). The suspension was incubated for 20min at 80°C and stored at -20°C.
+ Open protocol
+ Expand
2

Preparation of Phosphate Buffered Saline

Check if the same lab product or an alternative is used in the 5 most similar protocols
Sodium chloride (NaCl), sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) and monobasic potassium phosphate (KH2PO4) were purchased from Sigma-Aldrich and used to prepare phosphate buffered saline (PBS, pH of 7.4) solution containing 131 mM NaCl, 5.1 mM Na2HPO4·2H2O, and 1.5 mM KH2PO4 with highly purified water (18.2 MOhm cm). The PBS solution was degassed before used as receptor and donor solution in the Franz cells.
+ Open protocol
+ Expand
3

Botulinum Neurotoxin Preparation and Validation

Check if the same lab product or an alternative is used in the 5 most similar protocols
All chemicals were purchased from Sigma‐Aldrich (St Louis, MO) and solubilized in distilled water.
Natural Botulinum toxins A and B were purchased from List Biologicals Laboratories, Campbell, CA. They were prepared as stock solutions (666 nmol/L) in PBS (Gibco, Invitrogen, Cergy‐Pontoise, France); 1 mmol/L KH2PO4, 155 mmol/L NaCl and 3 mmol/L Na2HPO4) supplemented with 1 mg/mL BSA (Sigma‐Aldrich) and stored at −80°C. Dilutions were prepared as needed, in Krebs–Henseleit buffer (KHB; mmol/L: NaCl, 118; KCl, 4.7; CaCl2, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; glucose, 11) supplemented with 0.5% gelatin type A (Sigma‐Aldrich).
The activity of the toxins was confirmed in cell‐free assays using BoTest® (Botulinum Neurotoxin Detection Kit; BioSentinel, Madison, WI) and in cell‐based assays using rat spinal cord neuronal cultures.
+ Open protocol
+ Expand
4

Isolated Basilar Artery Contractility Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
During the isolated heart experiments, after thoracotomies performed under ketamine/xylazine anaesthesia, the brain was removed and placed into a silicone containing petri dish, filled with 0–4 °C Krebs solution (composition in mmol: 110 NaCl, 5.0 KCl, 1.0 MgSO4, 1.0 KH2PO4, 5.0 glucose and 24.0 NaHCO3, obtained from Sigma-Aldrich, St. Louis, MO, USA). The solution was equilibrated previously with a gaseous mixture of 5% CO2, 10% O2 and 85% N2 at pH 7.4. Basilar arteries were isolated with microsurgical tools (Fine Science Tools GmbH, Heidelberg, Germany). The arteries were equally cut into 4 mm long rings, which were then mounted in an isometric contraction measurement system (DMT-510, Danish Myotechnology, Aarhus, Denmark). The Ca2+-free Krebs solution was changed to Ca2+-containing one (composition in mM: 110 NaCl, 2.5 CaCl2, 5.0 KCl, 1.0 MgSO4, 1.0 KH2PO4, 5.0 glucose and 24.0 NaHCO3, obtained from Sigma-Aldrich, St. Louis, MO, USA). Before every experiment, a normalization protocol was performed, by stretching the preparations with 1.5 mN force, which was increasing evenly every 15 s until the calculated intraluminar pressure reached 13.4 kPa. The experiments were then performed at this stretch level. Contractile responses for KCl (6–66 mM), serotonine (1 nM–10 μM) and angiotensin II (1 nM–100 μM) have been recorded.
+ Open protocol
+ Expand
5

Artificial Saliva Buffers Preparation

Check if the same lab product or an alternative is used in the 5 most similar protocols
Artificial saliva (demineralising and remineralising buffers) were prepared according to Ten Cate et al [16] :
1-Demineralising buffer (AS4): 2.0 litres AS4 was prepared by dissolving 0.4411g of CaCl2.2H2O, 0.245g KH2PO4 (all Sigma-Aldrich) in 800ml of deionized water, and adding 5.72ml acetic acid (Sigma-Aldrich). The pH was adjusted to 4, by adding 0.5M KOH (Sigma-Aldrich), and deionized water was then added to make up a total volume of 2.0. The solution was stored in a fridge at 2°C. 2-Remineralising buffer (AS7): 2.0 litres AS7 was prepared by dissolving 0.4411g CaCl2.2H2O, 0.245g KH2PO4, 9.532 Hepes and 19.386g KCL (all Sigma-Aldrich) in 800ml of deionized water. The pH was adjusted to 7, by adding 0.5M KOH (Sigma-Aldrich) and deionized water was then added to make up a total volume of 2.0 litres. The solution was stored in a fridge at 2°C.
+ Open protocol
+ Expand
6

Peptide Fractionation and Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
The labeled peptides were reconstituted in 10% formic acid prior to fractionation using strong cation exchange (SCX) as described previously (Helbig et al, 2010 (link)) for the protein expression levels analysis. The SCX system consisted of an Agilent 1100 HPLC system (Agilent Technologies, Waldbronn, Germany) with two C18 Opti-Lynx (Optimized Technologies, OR) trapping cartridges and a polysulfoethyl A SCX column (PolyLC, Columbia, MD; 200 mm × 2.1 mm inner diameter, 5 μm, 200-A). The peptides were dissolved in 10% FA and loaded onto the trap columns at 100 μl/min and subsequently eluted onto the SCX column with 80% acetonitrile (ACN; Biosolve, the Netherlands) and 0.05% FA. SCX buffer A was made of 5 mM KH2PO4 (Merck, Germany), 30% ACN and 0.05% FA, pH 2.7; SCX buffer B consisted of 350 mM KCl (Merck, Germany), 5 mM KH2PO4, 30% ACN and 0.05% FA, pH 2.7. The gradient was performed as follows: 0% B for 10 min, 0–85% B in 35 min, 85–100% B in 6 min and 100% B for 4 min. After injection of 200 μg of labeled lysate, a total of 45 fractions were collected, dried in a vacuum centrifuge and stored at −80°C.
+ Open protocol
+ Expand
7

Isolation of Ovine Neutrophils

Check if the same lab product or an alternative is used in the 5 most similar protocols
Healthy adult Suffolk sheep (n = 6) were bled by puncture of the jugular vein and peripheral blood was collected in BD Vacutainer® heparin tubes (Franklin Lakes, USA). Approximately 20 mL of heparinized blood were diluted in 20 mL sterile PBS with 0.02% EDTA (Sigma-Aldrich, St. Louis, USA), layered on top of 12 mL Biocoll® separating solution (density = 1.077 g/L; Sigma-Aldrich) and centrifuged (800 × g, 45 min). After removal of plasma and peripheral blood mononuclear cells (PBMC), the cell pellet was suspended in sterile Hanks’ Balanced Salt Solution 1X (HBSS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for subsequent lysis of ovine erythrocytes by using lysis buffer solution (0.3 mM NaH2PO4, 0.4 mM KH2PO4, all Merck, Burlington, Massachusetts, USA) for 1 min at room temperature (RT). Then, a hypertonic buffer [0.3 mM NaH2PO4, 0.4 mM KH2PO4, 0.136 mM NaCl (all Merck)] and HBSS were added to the solution and centrifuged at 600 × g for 10 min. This process was repeated twice. Finally, the pellet was washed in sterile HBSS solution at 600×g for 10 min two times. PMN were re-suspended in sterile HBSS medium (Gibco), counted in a Neubauer haemocytometer chamber, and left on ice to rest (30 min) before use.
+ Open protocol
+ Expand
8

Anaerobic Cultivation of Sulfate-Reducing Bacteria

Check if the same lab product or an alternative is used in the 5 most similar protocols
Inside the anaerobic chamber (Bactron VI, Shellab, Sheldon Manufacturing, Incorporation using a mixed gas of 5% hydrogen, 5% carbon dioxide, and 90% nitrogen) at 38 °C a volume of 100 μL of mix culture of SRB was inoculated and enriched in culture standard medium. The Postgate standard medium was used for the growth of the SRB; for the maintenance a sulfate poor Postgate medium, agar was used and it is referred as the modified Postgate medium. Sodium lactate was used as a carbon source. Blackening of the medium due to the formation of iron sulfides was indicative of positive growth.
The Postgate standard medium contained per L: agar, 2.0 g (Difico); KH2PO4, 0.5 g (Merk); NH4Cl, 1.0 g (J.T. Backer); Na2SO4, 1.0 g (Merk); CaCl2, 1.0 g (Vetec); MgCl2·6H2O, 1.83 g (Merk); yeast extract, 1.0 g (Merk); ascorbic acid, 0.1 g (Merk); sodium thioglycolate, 0.013 g (Vetec); sodium citrate, 6.38 g (Synth); sodium lactate 1.75 mL; NaCl (Quemis) 3.5%, resazurin, 2.0 mL (Vetec) 0.025% P/V, FeSO4·7H2O, 0.5 g (Merk). All the components were dissolved in distilled water and the pH was adjusted to 7.5–8.0 using 5 M HCl. After this, the solution was homogenized by agitation and later sterilized at 121 °C for 30 min.
+ Open protocol
+ Expand
9

4C-seq Analysis of Zebrafish Pancreas

Check if the same lab product or an alternative is used in the 5 most similar protocols
4C-seq was performed as previously described88 (link), with minor alterations. Whole pancreas was dissected from 6 to 12 adult zebrafish (7–15 × 106 cells; both genders and with 12–24 months), kept on ice in PBS [137 mM NaCl (#S/3161/60, Fisher Chemical), 2.7 mM KCl (#2676.298, VWR), 10 mM NaHPO4 (#1.06342.0250, Merk), and 1.8 mM KH2PO4 (#1.06585.1000, Merk)] with 1x Complete Proteinase Inhibitor (#11697498001, Roche), fixed in 2% formaldehyde (#F1635-500ML, Sigma-Aldrich) for 10 min, and stored at −80 °C. Cell lysis was performed on ice, with a 15 mL Tenbroeck Homogenizer, not exceeding 10 min. Ligation was performed with 60U T4 DNA Ligase (#EL0012, ThermoFisher Scientific). The restriction enzymes used were DpnII (#R0543M, NEB) and Csp6I (#ER0211, ThermoFisher Scientific) for the first and second cuts, respectively. Chromatin was purified by Amicon Ultra-15 Centrifugal Filter Device (#UFC901024, Milipore). 4 C libraries were prepared for Illumina sequencing by the Expand Long Template Polymerase (#11759060001, Roche) with primers targeting the TSSs of each gene and including Illumina adapters (Supplementary Dataset 4c). Final PCR products were purified with the High Pure PCR Product Purification Kit (#11796828001, Roche) and AMPure XP PCR purification kit (#B37419AB, Agencourt AMPure XP).
+ Open protocol
+ Expand
10

Isolation of Fluorescent Zebrafish Islets

Check if the same lab product or an alternative is used in the 5 most similar protocols
The whole pancreases were dissected from double transgenic adult zebrafish [Tg(ins:GFP, ela:mCherry), Tg(ins:GFP, gcga:mCherry), and Tg(ins:GFP, sst:mCherry)] and fixed using 4% formaldehyde (#F1635, Sigma-Aldrich) in 1xPBS [137 mM NaCl (#S/3161/60, Fisher Chemical), 2.7 mM KCl (#2676.298, VWR), 10 mM NaHPO4 (#1.06342.0250, Merk), and 1.8 mM KH2PO4 (#1.06585.1000, Merk)]. Cells were dissociated, on ice, using a 15 mL Dounce homogenizer in 1 mL of ice-cold sort buffer [1% EDTA (#20301.290, VWR), 2 mM HEPES (#83264, Sigma-Aldrich) pH 7.0 in 1xPBS), and then passed through a 40-μm cell strainer. Immediately following dissociation, the mCherry and GFP fluorescence were analysed on a BD FACS-ARIATM II cell sorter (BD Biosciences).
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!