Pancreatic islets were isolated by collagenase digestion (0.7 mg/ml) (Sigma-Aldrich Chemie GmbH) and discontinuous Ficoll density gradient. Single-cell suspensions of pancreatic islets and lymphoid tissues were prepared using 70 μm cell strainers (Becton Dickinson, San Diego, CA, USA) and Hank’s buffer [1 x HBSS, 5% (v/v) FCS, 10mM HEPES; all Invitrogen]. Single cell suspensions from spleen were additionally subjected to red blood cell lysis (erythrocyte lysis buffer EL, Qiagen). Peripheral blood mononuclear cells (PBMCs) were obtained by retro-orbital sinus puncture [PBS supplemented with 10% (v/v) Heparin (Biochrom AG, Berlin, Germany)] and Ficoll (VWR, Darmstadt, Germany) gradient centrifugation. mAbs to CD3 (145-2C11), CD4 (RM4-5, GK1.5), CD8 (53–6.7), CD25 (PC61, 7D4), CD44 (IM7), CD62L (MEL-14), Vβ-4 (KT4), and CD49b (R1-2) were purchased from eBioscience (Frankfurt, Germany) or BD (Heidelberg). The samples were analyzed using a LSRII or sorted on a FACS Aria (all BD). Data were analyzed with the FlowJo software (Tree Star).
Ficoll
Manufactured by Avantor
Sourced in Germany, United States
Ficoll is a non-ionic, highly branched, and hydrophilic polysaccharide. It is commonly used in cell separation and purification procedures, particularly for the isolation of mononuclear cells from whole blood.
Automatically generated - may contain errors
Lab products found in correlation
70 μm cell strainer, by BD (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Ficoll, by Merck Group (1 mentions)
Heparin, by Harvard Bioscience (1 mentions)
Hank s buffer, by Thermo Fisher Scientific (1 mentions)
Erythrocyte lysis buffer el, by Qiagen (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Cryovials, by Avantor (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Tissue culture plastic, by BD (1 mentions)
Tryptone soya broth (tsb), by Thermo Fisher Scientific (1 mentions)
Catalase, by Merck Group (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
Uric acid, by Merck Group (1 mentions)
L gsh, by Merck Group (1 mentions)
Dextran, by Avantor (1 mentions)
4 abah, by Merck Group (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
3 protocols using ficoll
1
Isolation and Analysis of Murine Pancreatic Islets
2
Neutrophil Isolation from Whole Blood
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Following the aliquoting of 0.25–0.5 mL whole blood for neutrophil isolation, remaining blood volumes were diluted 1:2 with room temperature RPMI. Each diluted sample was then added carefully to a SepMate tube (STEMCELL Technologies, Cat# 85450 or 85,415) that had been prefilled with 15 mL Ficoll (VWR, Cat# 21008-918). Samples were spun at 1200 xg for 20 min at 20°C with maximum acceleration and the brake on. After centrifugation, the plasma layer was transferred into a clean conical tube and spun at 1000 xg for 5 min at 4°C to pellet any remaining cell debris. Without disturbing the pellet, each sample was aliquoted into 1.5 mL Cryovials (VWR, Cat# 66008-710) and frozen at −80°C until analysis.
3
Quantifying Neutrophil Oxidative Stress
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Dextran and Ficoll were purchased from VWR International (Radnor, PA, USA). Catalase, cytochrome C, H 2 O 2 , PMA, SOD, Triton X-100, 4-ABAH, DPI, L-GSH, NAC, uric acid, and DAPI were obtained from Sigma (St. Louis, MO, USA). Amplex UltraRed, APF, CellEvent Caspase-3/7 Detection Reagent Kit, CM-H 2 DCF-DA, DHR123, PrestoBlue resazurin, goat anti-rabbit Alexa Fluor 488, and Sytox Green and Orange were obtained from Thermo Fisher Scientific Life Sciences (Waltham, MA, USA). The polyclonal goat anti-human neutrophil elastase and rabbit anti-citrullinated histone H3 antibodies were purchased from Abcam (Cambridge, United Kingdom). Protein block was obtained from Dako (Carpinteria, CA, USA), and fixation buffer and antihuman IL-8 ELISA were purchased from BioLegend (San Diego, CA, USA). Tryptone soya broth was purchased from Oxoid (Thermo Scientific, Hampshire, United Kingdom), and Vectashield containing DAPI was obtained from Vector Laboratories (Burlingame, CA, USA). Tissue-culture plastic was obtained from Becton Dickinson (Franklin Lakes, NJ, USA), Nunc (Roskilde, Denmark), and Sarstedt (N ümbrecht, Germany).
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