About 5000 cells in 0.1 ml medium were seeded per well in a 96 well plate. After 24 hours of incubation, cells were treated with different concentrations of piperine and plates were incubated for 24, 48 and 72 hours. Cells were fixed using 10% tricholoroacetic acid (Sigma Aldrich Ltd.) and incubated for 1 hour at 4°C. Subsequently, cells were stained with 0.5% Sulforhodamine B solution and the absorbance were measured at 570 nm using a plate reader (BioTek Instruments, Winooski, VT) as described by us previously [12] (link), [13] (link).
Tricholoroacetic acid
Manufactured by Merck Group
Sourced in United States
Trichloroacetic acid is a colorless crystalline compound commonly used as a chemical reagent in laboratory settings. Its core function is as a protein precipitating agent, enabling the isolation and purification of proteins from samples.
Automatically generated - may contain errors
Lab products found in correlation
Thiobarbituric acid, by Merck Group (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Toluene, by Merck Group (1 mentions)
Proline, by Merck Group (1 mentions)
Ninhydrin, by Merck Group (1 mentions)
Cordycepin, by Merck Group (1 mentions)
Ethyl acetate, by Junsei (1 mentions)
Sulfosalicylic acid, by Merck Group (1 mentions)
Folin ciocalteu s phenol, by Merck Group (1 mentions)
Chloroform, by Kanto Chemical (1 mentions)
Hexane, by Kanto Chemical (1 mentions)
Methanol plus, by Junsei (1 mentions)
Acetone, by Kanto Chemical (1 mentions)
Sodium carbonate, by Kanto Chemical (1 mentions)
Methanol, by Junsei (1 mentions)
Sodium cholate, by Merck Group (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Tribarbituric acid, by Merck Group (1 mentions)
9 protocols using tricholoroacetic acid
1
Piperine Cytotoxicity Evaluation in Cell Cultures
2
Cordycepin-Based Antioxidant Assessment
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Cordycepin, proline, Folin-Ciocalteu’s phenol, ninhydrin, glacial acetic acid, thiobarbituric acid (TBA), tricholoroacetic acid (TCA), toluene and sulfosalicylic acid were bought from Sigma-Aldrich Japan K.K., Tokyo, Japan. Ethyl acetate, methanol, and methanol plus were provided from Junsei Chemical Co., Ltd., Tokyo, Japan. Hexane, chloroform, acetone, sodium carbonate and aluminum (III) chloride hexahydrate were obtained by Kanto Chemical Co. Inc., Tokyo, Japan.
3
Concentrating Cell Supernatants using TCA
4
Plasma MDA Quantification by TBARS Assay
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Plasma concentration of MDA was determined using a TBARS assay. Briefly, plasma samples and standards (1,1,3,3-tetraethoxy propane, Sigma) were mixed with 0.25 N, HCl (Sigma), 20% tricholoro acetic acid (Sigma) and 0.8% tribarbituric acid (Sigma) incubated at 90 °C for 60 min, cold in ice, and centrifuged at 4000 rpm for 10 min. Then, 200 μl of prepared solutions was added to each well, and the absorbance of well-plate was read at 532 nm using a microplate reader (Biotek, USA) [48 (link)].
5
Analyzing Bacterial T3SS Protein Secretion
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Bacteria were grown overnight under T3SS inducing conditions in LB media containing 5 mM ethylene glycol-bis(2-aminoethylether) (EGTA) (Sigma-Aldrich, St. Louis, MO, USA). The next day the suspension was diluted, and compounds were added to a final concentration of 50 μM in a 50 mL tube. After another 4 h of co-incubation the supernatant was separated via centrifugation and filtered through a 0.45 μm-pore-size low protein-binding filter (ThermoFisher, Scientific, Waltham, MA, USA). Subsequently proteins were precipitated by tricholoroacetic acid (Sigma Aldrich, St. Louis, MO, USA), washed and analyzed by SDS-PAGE using Instant Blue Coomassie dye (Expedeon, Heidelberg, Germany).
6
Colorimetric Assay for Protease Activity
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The colorimetric assay to measure protease activities was adapted from [50 (link),51 (link)]. In short, casein (Sigma-Aldrich, cat. no. C7078-500G) was digested to release tyrosine. After stopping the digestion with tricholoroacetic acid (Sigma-Aldrich, cat. no. T0699-100ML), released tyrosine was measured by absorption at 660 nm using Folin & Ciocalteu's Phenol reagent (Sigma-Aldrich, cat. no. F9252-100ML). Enzymes were tested at their respective optimal working temperature and digestion buffer for 10 min. Concentration ranges were 0.2–1.0 U ml−1 for proteinase K, 0.2–1.0, 10 U ml−1 and 50 U ml−1 for Zymolyase and β-glucoronidase and 0.02–0.1 U ml−1 for lysing enzyme. Controls with 20 mM of the protease inhibitor phenylmethylsulfonylfluorid (PMSF, ThermoFisher Scientific, cat. no. 36978) were conducted for proteinase K and lysing enzyme and a standard curve of free l -tyrosine (Sigma-Aldrich, cat. no. T3754-50G) was measured.
7
Cerastes cerastes Venom Extraction and Characterization
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Cerastes cerastes (Cc) venom was milked from adult vipers which collected from El-Faiyum, Egypt. Directly after extraction, the venom was pooled, divided in aliquots, lyophilized, and stored at –20 °C. Azocasein, tricholoroacetic acid, Tris base, acrylamide, bis-acrylamide, fibrinogen, gelatin, coomassie brilliant blue R-250, l -Leucine and o-phenylenediamine (OPD) were obtained from sigma. ALT and AST diagnostic kits were purchased from Biodiagnostic Co., Egypt. All other chemicals and reagents were of analytical grade. The buffers were prepared according to Gomorie15 and the final pH was checked by pH meter (Hanna, pH 211 Microprocessor pH meter).
8
Analytical Reagents and Standard Compounds
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All chemicals and reagents used in current study were of analytical grade. Solvents i.e. n-hexane, ethyl acetate, methanol and dimethylsulfoxide (DMSO) were purchased from Merck (Darmstadt, Germany). Folin-Ciocalteu reagent and DPPH were purchased from Sigma–Aldrich (Steinheim, Germany). Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous chloride, sodium hydroxide, aluminum chloride, ascorbic acid, quercetin, gallic acid, rutin, caffeic acid, kaempferol, myricetin and (+)-catechin were acquired from Sigma–Aldrich (Steinheim, Germany). Tween 80, thiobarbituric acid, tricholoroacetic acid, ferric chloride and phenazine methosulphate were acquired from Sigma (Chemicals Co. St. Louis, USA). Sodium carbonate, sulphuric acid, hydrogen peroxide, potassium ferricyanide, sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Merck KGaA (Darmstadt, Germany).
9
Optimized Immunofluorescence Staining and Imaging of Spinal Cord
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Immunofluorescence staining for CD3 and B220 was performed as previously reported (6 (link)). Intermittent background labeling from anti-CD3 staining was noted, but consistently within the gray matter and not in a pattern confounding for cellular labeling of T cells. Background immunofluorescence was a result of anti-CD3, as control staining with secondary antibody only never resulted in excessive background signal. Staining for additional markers, including VCAM-1, Ly-6G, podoplanin, and Ki-67 is detailed in SI Appendix . Optical clearing of spinal cords was initiated by perfusing mice with 25 mL ice-cold phosphate buffered saline followed by 20 mL 4% paraformaldehyde (Sigma-Aldrich). CNS tissue was extracted while still encased in the skull and vertebral column, then fixed in 4% paraformaldehyde for more than 12 h. Spinal cord vertebrae were decalcified in 6% tricholoroacetic acid (Sigma-Aldrich) for 5 d and washed with PBS prior to optical clearing using iDISCO protocol (36 (link)). Additional details including confocal imaging are provided in SI Appendix .
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