Cells were centrifuged in 210 for 5 min and seeded to tissues culture flasks seeing that required (T25 flasks for regimen maintenance and T75 flasks for planning of co-cultures)

Cells were centrifuged in 210 for 5 min and seeded to tissues culture flasks seeing that required (T25 flasks for regimen maintenance and T75 flasks for planning of co-cultures). for 5 min, aspirate the resuspend and supernatant in finish lifestyle medium. Count number cells (3T3Neo and 3T3CD40L) and calculate the mandatory level of cell suspension system. G3581) Mobile DNA Fragmentation ELISA package (for 500 exams) (Roche Diagnostics, catalog amount: 11585045001) Cytotox-GloTM cytotoxicity assay (5 x 10 ml) (Promega, catalog amount: G9291) Sensolyte? Homogeneous AFC Caspase-3/7 Assay Lonaprisan package (Cambridge Bioscience, catalog amount: “type”:”entrez-protein”,”attrs”:”text”:”ANA71114″,”term_id”:”1022531083″,”term_text”:”ANA71114″ANA71114) CM-H2DCFDA (chloromethyl derivative of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate) (Thermo Fisher Scientific, catalog amount: C6827) Glycerol (Fisher Scientific, catalog amount: Lonaprisan 10795711) Sodium dodecyl sulphate (SDS) (Thermo Fisher Scientific, InvitrogenTM, catalog amount: NP0002) Tris-HCl, natural powder, 1 KG (Melford, catalog amount: T1513) Sodium fluoride (Acros Organics, catalog amount: 424325000) Sodium pyrophosphate tetrabasic (Sigma-Aldrich, catalog amount: P8010-500G) Sodium orthovanadate (Sigma-Aldrich, catalog amount: S6508-10G) Protease inhibitor (PI) cocktail (Merck, catalog amount: 535140-1) PI cocktail (New Britain Biolabs, catalog amount: 5872S) AQUAGUARD-1 alternative for disinfection of Rabbit polyclonal to KBTBD7 drinking water baths and CO2 incubators (Biological Sectors, catalog amount: 01-867-1B) (find Meals) 1x PBS (find Meals) PBS/EDTA alternative (see Meals) Freezing moderate (see Recipes) FACS buffer (see Recipes) 70% ethanol (see Recipes)CEthanol, Absolute (Fisher Scientific, catalogue number: E/0650DF/P17) DR medium (see Recipes) SSB buffer (see Recipes) Lysis buffer (see Recipes) Equipment Gilson Pipettes P1000 (200-1,000 l) P200 (50-200 l) P20 (2-20 l) P10 (1-10 l) Gilson REPETMAN electronic pipette 0.1-50 ml (Gilson, catalog number: F164503) Water bath (Memmert) Boiling water bath (Grant) -80 C freezer Sparkfree Refrigerator and Freezer (Labcold) Electrophoresis Power Supply Refrigerated centrifuge (PRISM R) Universal 320 benchtop centrifuge (Hettich Zentrifugen) Vortex mixer Ultrasonic Homogenizer Sonicator NuAire CellGard ES Biological Safety Cabinet (TripleRed) Iso Class 5 Nuaire Autoflow IR direct heat CO2 incubator with a HEPA filtration system at 37 C and 5% CO2 (TripleRed) OdysseyTM Infra-red Imaging system (Li-Cor) Guava EasyCyteTM Flow Cytometer (Millipore) Countess II Automated Cell Counter (Thermo Fisher Scientific, catalog number: AMQAX1000) EVOSTM XL Core Imaging System (Fisher Scientific) FLUOstar OPTIMA (BMG Labtech) Software MARS software (BMG Labtech), Version 2.0 Guava EasyCyte flow cytometry software (Millipore), Guavasoft Version 2.6 Image Studio Life, Version 4.0 Adobe Photoshop CS, Version 8.0 Procedure General cell culture Cell maintenance and passage Cells were routinely maintained at 37 C in a 5% CO2 humidified atmosphere provided by autoclaved ddH2O, supplemented with 1% AQUAGUARD-1 (see Recipes). All class II cabinet surfaces as well as CO2 incubators were routinely disinfected with either Mikrozid (weekly) or 70% ethanol (daily) solution. Cultured cells were routinely observed by phase contrast microscopy. All cell lines were maintained in T75 flasks with 12-14 ml medium or T25 flasks in 4-5 ml medium. All cell lines were sub-cultured every 2-3 days or when ~80-95% confluent. Epithelial cells were routinely passaged at 1:10 ratios and effector (3T3Neo and 3T3CD40L) cells at 1:6 ratios. For routine passaging, cells were collected by washing with a solution of 0.1% (w/v) EDTA Lonaprisan in phosphate buffered saline (EDTA/PBS) (see Recipes) for 5 min and then addition of Trypsin-EDTA solution, until cells detached from the culture flasks. Trypsin was inactivated by the addition of the respective serum-supplemented culture medium when cells were re-suspended, followed by centrifugation to aspirate medium and resuspension in fresh culture medium. Cultured cells were medium-changed every two days, unless otherwise stated. Lonaprisan 3T3 cells were harvested and passaged as with carcinoma cells with the exemption of a very short EDTA/PBS treatment, as extended periods risked rapid cell detachment. Cryopreservation and recovery For long-term storage, following growth and appropriate passage, cell banks and sub-banks were cryo-preserved in liquid nitrogen as described below. For cryopreservation of cell lines, cultures were collected by routine passaging and precipitated by centrifugation. The cell pellet was re-suspended in Freezing medium (see Recipes) at a cell density not less than 1 x 106 cells/ml. Cells were aliquoted in a total of 1-1.5 ml to polypropylene cryovials and then transferred to an ice-cold Mr Frosty freezing container containing 250 ml of isopropanol to control the cooling rate to 1 1 C per minute. Cells were then placed within a -80 C freezer for 4-6 h prior to transfer to liquid nitrogen. Cells were recovered by thawing rapidly at 37 C, before 5-10 ml of pre-warmed growth medium was added. Cells were centrifuged at 210 for 5 min and then seeded to tissue culture flasks as required (T25 flasks for routine maintenance and T75 flasks for preparation of co-cultures). for 5 min, aspirate the supernatant and resuspend in complete culture medium. Count cells (3T3Neo and 3T3CD40L) and calculate the required volume of cell suspension. Add 100 l containing 1 x 104 cells per well in 96 well plates (for cytotoxicity assays), 6 x 104 cells per well in 24 well plates (for supernatant collection), 3 x 105 cells per well in 6 well plates, or.