The percentage of apoptotic cells was established by counting in excess of cells in at the least three separate randomly chosen microscopic fields Annexin V staining for detection of apoptotic cells Flow cytometric analsis for detection of apoptotic cell population was carried out annexin V staining using with annexin VFITC apoptosis detection kit as per manufacturer?s instruction. Tumor cells had been washed thrice with PBS and resuspended in binding buffer containing annexin V FITC reagent. Immediately after incubation at space temperature for min, the cells were incubated in PI RNase for min, observed under fluorescence microscope and analyzed by flow cytometer for apoptotic cells. Bone marrow colony forming assay Bone marrow colonies were prepared as outlined by a process described earlier implementing culture medium containing methylcellulose . Briefly, BMC had been suspended in the mixture containing . methylcellulose with FCS and LCM. The mixture was gently vortexed, plated within a mm plastic culture dish and incubated at C in the humidified atmosphere of CO in air. Bone marrow colonies were counted just after ten days of incubation. An aggregate of in excess of cells was counted like a single colony forming unit .
Colonies of different kinds have been identified around the basis of their morphological qualities. These with macrophage like morphology had been designated as CFU M, granulocyte macrophage drug library selleck chemicals morphology as CFU GM and granulocyte morphology as CFU G. Culture and isolation of bone marrow derived macrophages BMDM were obtained as described previously . Briefly, mice were killed by cervical dislocation and BMC have been flushed from femoral shafts with chilled serum absolutely free medium. A single cell suspension of BMC was ready and incubated in plastic tissue culture flasks for h to clear away adherent bone marrow macrophages. The non adherent BMC were harvested and incubated for days in medium containing LCM . Immediately after incubation for days, viable adherent BMDM were detached utilizing a cell scrapper as well as BMDM quantity was determined. Flow cytometric examination was carried out to examine the expression of F and CDc markers making use of flow cytometer following producer?s instruction.
After washing cells had been plated within a flat bottom properly plastic culture plate at a cell density of cells very well. Methotrexate Just after h of incubation the cultures were vigorously shaken and washed with warm medium to take away non adherent cells. A lot more than of your adherent cells were favourable for phagocytic capacity, non exact esterase staining and expression of macrophage marker F with typical macrophage morphology. The BMDM as a result obtained have been even more incubated for h in medium alone or containing LPS plus IFN g . Soon after h of incubation the cellfree culture supernatant was harvested for assay of IL , IL and TNF a production as well as the cells had been employed for other estimations described beneath Cytotoxicity assay Macrophage mediated tumor cytotoxicity was assayed by measuring the killing of target DL cells as described earlier with some modifications.