Platelets were isolated from Wistar rat as Musaji described [8 (link)]. In brief, blood was collected from jugular vein of Wistar rats with 1/6 volume ACD (citrate-dextrose solution, Sigma-Aldrich, Bornem, Belgium). Platelet-rich supernatant was prepared by successive centrifugations at 10°C for 10 minutes at 200g. Platelets were pelleted from this supernatant by additional centrifugation at 10°C for 12 minutes at 1700g and washed as appropriate. CBA mice were divided into three groups which are control and ITP model group with/without indirubin treatment respectively (n = 10). The two group mice for ITP model were first administered with rat platelets 108 in 0.5 ml saline intraperitoneally, followed by an injection of 0.5×108 platelets weekly. Control mice were injected with saline alone. Indirubin was obtained from Cal Biochem and diluted in the vehicle containing sodium CMC (C-5013 high viscosity, Sigma-Aldrich). Experimental ITP mice received i.p. administration of 50 mg/kg indirubin daily one week before immunization. Pure ITP mice were injected with CMC only.
Citrate dextrose solution
Manufactured by Merck Group
Sourced in United States
Citrate-dextrose solution is a laboratory product used as an anticoagulant in the collection and storage of blood samples. It functions by inhibiting the coagulation process, allowing the blood to remain in a liquid state during handling and analysis.
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Lab products found in correlation
G46 2.6 fitc, by BD (2 mentions)
Facs fusion cell sorter, by BD (2 mentions)
Lsrfortessa cell analyzer, by BD (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Prostaglandin e1, by Merck Group (1 mentions)
Polypropylene tube, by Corning (1 mentions)
Complete rpmi 1640 medium, by Merck Group (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Polymorphprep kit, by Axis-Shield (1 mentions)
Proteominer protein enrichment kit, by Bio-Rad (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Fetal bovin serum, by Thermo Fisher Scientific (1 mentions)
L glutamin, by Thermo Fisher Scientific (1 mentions)
Dulbeco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Carboxymethylcellulose cmc, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Daejung Chemicals (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
14 protocols using citrate dextrose solution
1
Induction and Treatment of Immune Thrombocytopenia in Mice
2
Murine Anti-MHC I Lung Injury Assessment
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Mouse temperatures were measured using a rectal probe and digital thermometer (Bioseb, Pinellas Park, USA), prior to anti-MHC I injection and then every 10 min for 2 h, or until death. Mouse survival rates were evaluated for 2 h and at 48 h following treatment. First set of mice were used to calculate wet lung to body weight ratio. Other set of mice were used to obtain bronchial lavage fluid (BAL) through a tracheotomy with a 25-gauge catheter before the pneumonectomy. Lavage was conducted using a 1 ml injection of cold PBS flushed back three times. BAL cells were centrifuged at 491 g for 10 min and resuspended in PBS. BAL platelets were enumerated using an MS4™ Hematology Analyzer (Melet Schloesing Laboratoires, Osny, France). BAL total proteins were evaluated by Bradford assay. Peripheral-blood cell number and MPV were determined using the MS4™. Intracardiac puncture was conducted with a 25-gauge needle and anticoagulant citrate-dextrose solution (Sigma Aldrich, Saint-Louis, USA). Tail vein injections used a 30-gauge needle. Plasma samples were collected and stored. The BAL platelet ratio was calculated as follows:
3
Platelet Caspase-3/7 Activity Modulation
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Blood samples were collected from the retro-orbital venous plexus into polypropylene tubes (Axygen) containing 10% citrate-dextrose solution (Sigma). Platelet rich plasma was obtained by centrifugation of the blood samples at 200 g for 10 minutes. The resultant platelet rich plasma was then incubated with 0.1 μg/mL Prostaglandin E1 (Sigma) in PBS for 10 minutes at room temperature and concentrated by centrifugation at 2250 g for 15 minutes. The concentrated platelets were cultured ex vivo for 2 hr in the presence or absence of anti-CD41 antibody before subjecting to LLLT or sham light and measurement of caspase-3/7 activity by Caspase-Glo. To measure caspase-3 activation in platelets in vivo, platelets were prepared from mice after treated with or without anti-CD41 antibody for 2 days, along with two doses of LLLT or sham light. Caspase-3 activation was assayed by FAM-DEVD-FMK staining.
4
Platelet Dynamics in SIV-Infected Macaques
5
Analyzing IL-10 Production in B Cells
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PBMCs were cryopreserved in 10% dimethyl sulphoxide and stored at −80°C in liquid nitrogen. For the experiments, PBMCs were thawed directly in 37°C heated RPMI 1640 complete medium (Sigma‐Aldrich, St. Louis, MO, USA) supplemented with 10% inactivated human AB serum and 1% penicillin/streptomycin. Then, the cells were washed in 10X PBS supplemented with 5% citrate‐dextrose solution (Sigma‐Aldrich), resuspended in fresh complete RPMI medium and plated in a 96‐well U‐bottom plate at a concentration of 1 × 106 cells mL‐1. Analysis of IL‐10 production from B cells was evaluated by two in vitro methods upon stimulation with CpG (cytosine guanine dinucleotide [CpG] ± CD40L), as previously described.38 After 24 h of incubation, cells were stained for CD19 surface marker and permeabilised IL‐10 was detected by anti‐IL‐10 isotype‐matched control. In the same assay, one well with no activated cells was separated for evaluation of the frequency of B‐cell subsets using the following anti‐human monoclonal antibodies (mAbs): CD19 (HIB19), CD27 (L128), CD38 (HIT2), immunoglobulin M (IgM) (G20‐127) and immunoglobulin D (IgD) (IA6‐2) from BD Pharmingen (San Diego). Cells were acquired in a FACSAria II flow cytometer (Becton‐Dickinson), and data were analysed by the Flow Jo (TreeStar) software.
6
Cell Culture and Neutrophil Isolation
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RPMI 2650 (ATCC CCL30) and HaCaT cells (63 (link)) were maintained in RPMI 1640 and Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), respectively. Neutrophils were isolated from healthy donors (with use and procedures approved by the National Taiwan University [IRB 201711091RIND]) using a PolyMorphPrep kit (Axis-Shield, Oslo, Norway) per the manufacturer’s instructions, while platelets were prepared by centrifugation from blood anticoagulated with citrate-dextrose solution (Sigma) as previously described (64 (link)).
7
Isolation and Characterization of Murine Platelets
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PLTs were isolated from the whole blood of the mice (19 (link)). Briefly, 6:1 of citrate-dextrose solution (Sigma-Aldrich; Merck KGaA) and whole blood were gently mixed and centrifuged at 280 x g for 15 min at room temperature. The PLT-rich plasma (PRP) was obtained by another centrifugation at 400 x g for 5 min at room temperature. To obtain PLT pellets, PRP was further centrifuged at 1,100 x g for 10 min at room temperature and the PLT pellets were suspended in PBS for immediate use. In the indicated experiments, PLTs were stained with CD41 antibody and visualized under a fluorescence microscope or analyzed by flow cytometry.
8
Enrichment of Low-Abundant Plasma Proteins
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Venous blood from five AIH mice and five normal mice was collected from the angular vein using the anticoagulant tubes, which were pretreated with citrate-dextrose solution (Sigma-Aldrich, St. Louis, MO USA) and then centrifuged at 500g for 10 min to obtain the supernatant (plasma). To obtain and concentrate as much of the low-abundant proteins as possible, the samples were equalized using the ProteoMiner Protein Enrichment Kit (Bio-rad laboratories, Hercules, CA, USA), according to the manufacturer’s instructions. Each column was loaded with the samples, which were first passed through a 0.22-μm-filter. No bead agglomeration was observed. The proteins were desorbed using a two-step elution process. First, the beads were treated twice with 100 μL of the kit elution reagent (4 M urea, 1% (w/v) CHAPS, 5% (v/v) acetic acid) for 15 min. Then, 100 μL of 6 M guanidine-HCl (pH 6.0) was added twice, followed by incubation for 15 min. Finally, four elution fractions from each column were pooled and stored at − 80 °C for further analysis.
9
Isolation and Characterization of Melanoma Cells
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Melanoma cells were identified and sorted by flow cytometry as described previously (15 (link), 20 (link)). All antibody staining was performed for 20 min on ice, followed by washing with HBSS and centrifugation at 200 × g for 5 min. Cells were stained with directly conjugated antibodies against mouse CD45 (APC, eBiosciences), mouse CD31 (APC, Biolegend), mouse Ter119 (APC, eBiosciences), and human HLA-ABC (G46-2.6-FITC, BD Biosciences). Human melanoma cells were isolated as cells that were positive for HLA and negative for mouse endothelial and hematopoietic markers. Cells were washed with staining medium and resuspended in DAPI (1 μg/mL; Sigma) to eliminate dead cells from sorts and analyses. Cells were examined on an LSRFortessa cell analyzer (Becton Dickinson) or sorted on a FACS Fusion Cell Sorter (Becton Dickinson). For analysis of circulating melanoma cells, blood was collected from mice by cardiac puncture with a syringe pretreated with citrate-dextrose solution (Sigma) when subcutaneous tumors reached 2.5 cm in diameter. Red blood cells were sedimented using Ficoll (Ficoll Paque Plus, GE Healthcare). The remaining cells were washed with HBSS (Invitrogen) before antibody staining and flow cytometry.
10
Comprehensive Biomaterial Characterization Protocol
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RG was supplied by CheonJiYang Co. (Seoul, Korea), and CS (MW 1,000-3,000) was purchased from Kitto Life Co. (Seoul, Korea). PGA (MW 50 kDa) and Fu extracted from Laminaria japonica were purchased from Bio Leaders Corp. (Daejeon, Korea) and Haewon Biotech Inc. (Seoul, Korea), respectively. Citrate dextrose solution, Hepes buffer (4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid), bovine serum albumin, and carboxymethyl cellulose (CMC) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Collagen was purchased from Chrono-Log Co. (Havertown, PA, USA).
For the cytotoxicity test, BHK 21 cell was purchased from Korean cell line bank (Seoul, Korea). Dulbeco's modified eagle's medium, L-glutamin, and fetal bovin serum were purchased from Gibco Invitrogen Co. (Grand Island, NY, USA). CMC and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide was purchased from Dae-jung Chemical Co. (Seoul, Korea). All other chemicals were reagent grade and were used without further purification.
For the cytotoxicity test, BHK 21 cell was purchased from Korean cell line bank (Seoul, Korea). Dulbeco's modified eagle's medium, L-glutamin, and fetal bovin serum were purchased from Gibco Invitrogen Co. (Grand Island, NY, USA). CMC and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide was purchased from Dae-jung Chemical Co. (Seoul, Korea). All other chemicals were reagent grade and were used without further purification.
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