Human Embryonic Kidney (HEF-293 Cell Lab Report

Human Embryonic Kidney(HEK) 293 cells were transiently transfected with pEGFP-N1(Clontech, Palo Alto, CA, encoding enhanced GFP as an indicator of transfection) and pEGFP-LeIF plasmids cloned at previous studies(Ghafarifar and et al, in press) by Calcium phosphate and non- tranfected cells was used as negative control. briefly, Hek-293 cells were grown at 37°C in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and 5% CO2. Log-phase cells were employed for transfection experiments. Briefly, 24 h before transfection, cells were trypsinated and spilited into a 6-well plate (about 3 × 104 cells/well). The cells reached 40-60% confluence the day of transfection. 2-3h prior to transfection, culture
This DNA-CaCl2 mixture (5µg DNA) was incubated at Room Temperature for 5 minutes. Afterwards, the CaCl2-DNA solution was added drop wise to 250μl of 2X HBS (50 mM HEPES and 1.5 mM Na2HPO4, 280 mM NaCl and 20 mM KCl, adjusted to pH 7.12) and the solution was incubated at room temperature for 15 minutes to form a homogeneous calcium phosphate -DNA precipitate. Before transfection, the DNA-Ca2+ - phosphate solution was mixed and added to HEK- 293 cells by dropping slowly. The plate was swirled to distribute the precipitate equally over the
Briefly, Spleens were removed in sterile condition and homogenized using base of a syringe in PBS. Then, the resuspansed solution was washed twice in 300g centrifuged for 10min. erythrocytes were lysed Using cold Ammonium-Chloride-Potassium (ACK) lysis buffer (0.15 M NH4Cl, 10 mM KHCO3 and 0.1 mM Na2EDTA) for 5 mints at room temperature. Lymphocytes were washed with PBS contained 2% FBS and resuspened in RPMI-1640 supplemented with 10% FBS (Gibco, BRL, Maryland, USA). Viability and count of the cells was evaluated with aid of 0.4% of trypan blue and a haemocytometer slide under an optical microscope . Then, 3 ×106 cells/well were seed into 24 - well plates in duplicate. lymphocytes were stimulated with 25 μg/ml Frozen and thawed (F/T) antigen with liquid Nitrogen and 37ºC water bath for 10 times and incubated at 37ºC and 5% CO2 humidified atmosphere for 72hrs. The supernatant was slowly removed and aliquoted in 300µl volumes in 0.5 ml vials and were kept at 70 ºC until cytokine assay. IL-4 and IFN-γ were measured by sandwich based ELISA kits (U-CyTech biosciences, Netherland) according to recommended procedure. All tests were performed in