Thymocyte irradiation

Biologic effects of non-excited fullerenes C60, that are revealed at the concentration range lower than 10 4 M, are mostly positive, but depend on the type of cells and the way of modification of fullerene C60 (Yamakoshi et al., 1994). As we have shown earlier, upon the presence of 10 6 M fullerenes C60 in incubation medium, resistance of erythrocytes to hemolysis is not altered, whilst at the concentration of 10 5 M fullerenes C60 the hemolysis rate is accelerated. Hemolytic effect was not revealed if fullerene C. at the concentration of 10 5 M was introduced to the con-tent of aminopropylaerosyl (i.e., upon the presence of fullerene C60-composite-l). Cytotoxic influence was not found if thymocytes and EAC cells were incubated with fullerenes C60 (10 5 M) or fullerene C60-containing composites for 24 h (Piylutska et al., 2006). That is why the study of the influence of irradiation on biologic activity of fullerenes C60 was carried out at their concentration of 10 5 M. 

The influence of photoexcited fullerene C60 on viability of thymocytes, EAC, and L1210 cells was studied after 24 h of incubation, considering the content of viable cells at incubation as 100% (Table 6.1). After irradiation of fullerenes C60 in the cell medium, significant decrease in the content of viable cells in the suspension of thymocytes was not registered, whilst the number of viable malignant cells decreased. Upon the presence of photoexcited fullerene C60 in incubation medium, the number of viable EAC cells decreased by 20%, and L1210 by 12%, while in the... 

Taking into account that ROS produced by irradiated fullerenes C60 may act only in the radius of their short diffusion existence, one may suppose that cytotoxic effect is determined by the interaction of fullerene C60 with the surface of cells and initiation of chain reactions of free radical peroxidation in membranes. That is why the influence of photoexcited fullerene C60 on the course of LPO process was studied and evaluated by the content of generated primary (diene conjugates) and final (Schiff bases) products. The content of diene conjugates in thymocytes was 17.7 4.2, inEAC cells was 21.1 1.3, andinL1210 was 12.8 3.1 nM/mg protein, and Schiff bases -56 7.9,46.5 4.5, and 36.6 4.6 rel. units/mg protein, respectively, and did not alter during 1 h incubation of the cells. 

The rate of MTT reduction was determined adding the agent to cell incubation medium on 0.5,2, and 5 h after irradiation of samples. In the case of non-irradiation of fiillerenes C60 in incubation medium, the indexes of total metabolic state of thymocytes, EAC, and L1210 cells did not alter compared to the control. 

Staurosporine, UCN-01, and UCN-02 (a weak PKC inhibitor) induced a concentration- and time-dependent increase in apoptosis, whereas neither CGP 41251, RO 31-8220, nor GF 109203X induced apoptosis in immature rat thymocytes. In the human cell line BM13674 the specific inhibition of PKA gave rise to significantly increased levels of apoptosis at postirradiation compared to values after radiation e3q>osure only. Calphostin C which caused 68% inhibition of PKC activity in irradiated cells, did not alter the level of radiation-induced apoptosis (Findik et al., 1995). 

Ohmori T, Arteaga C]L (1998) Protein kinase C epsilon translocation and phosphorylation by cis-diamminedichloroplatinum(II) (CDDP) potential role in CDDP-mediated cytotoxicity. Cell Growth Differ 9 345-353 Ojeda F, Guarda MI, Maldonado C, Folch H, Diehl H (1992) Role of protein kinase-C in thymocyte apoptosis induced by irradiation. Int J Radiat Biol 61 663-667 Oka M, Ogita K, Ando H, Horikawa T, Hayashibe K, Saito N, Kikkawa U, Ichihashi M (1996) Deletion of specific protein tdnase C subspecies in human melanoma cells. J Cell Physiol 167 406-412... 

Feeder cells for fusion cultures (see Note 6) essential for fusions employing rat myelomas. Quickly thaw irradiated rat fibroblasts, prepared as described in Note 6, just before commencing the cell fusion. Add the cells to 10 mL of serum-free DMEM, centrifuge, and wash once in serum-free DMEM Resuspend feeders in HAT medium just before addition of the fusion mixture. Alternatively, use thymocytes from spleen donors (mouse). 

Centrifuge for 3 min at 400g, and then resuspend the cells in 200 mL HAT selection medium, and add irradiated fibroblast or thymocyte feeder cells. Plate 2-mL aliquots into four 24-well plates or, if necessary, five 96-well plates (fusions with SP2/0 myeloma) (see Note 6). 

Prepare a suspension of mouse thymocytes or irradiated rat fibroblasts as feeders (5 x 105/20 mL of HT or DMEM containing 10% FCS)... 

The autoantibody production in autoimmune diseases may be attributed to the inability of Treg cells to control their synthesis. In an autoimmune model, T cells regulate the mechanisms through which B cells that were autoreactive to selfantigens do not produce autoantibodies, suggesting a role for suppressor T cells. The administration of irradiation, thymocytes, lymph nodes or spleen cells inhibits the production of autoantibodies, which is attributed to the suppressor T cells. 

Feeder cells for fusion cultures ( eNote 5. Essential for fusions employing rat myelomas) Three hours to one day before fusion, 2—4 x 10 rat fibroblasts in 10 mL of DMEM and contained in a 30 mL plastic universal are irradiated with about 30 Gy (3000 rad) of X- or T rays. Dilute with 90 mL of HT (3 h before fusion) or DMEM containing 10% FCS (1 d before fusion) and plate 1-mL aliquots into four 24-well plates or 0.2-mL aliquots into five 96-welI plates. Alternatively, use thymocytes from spleen donors (mouse). 

Prepare a suspension of mouse thymocytes (2.5 x 10 /mL of DMEM-10% FCS) and use direcdy or seed irradiated rat fibroblasts in DMEM-10% FCS at 5 X 10 cells per well into 96-well plates for use the next day. 

Bcl-2 expression prevents or delays apoptosis under a variety of circumstances. These include (a) withdrawal of the neurotrophins NGF, BDNF, and NT-3 from primary neuron cultures and from PC 12 cells (Garcia et al., 1992 Allsopp et al., 1993 Mah et al., 1993), (b) induction of apoptosis in thymocytes by glucocorticoids, irradiation, anti CD3-Ab (anti-TCR ab) and Ca + ionophores (Sentman et al., 1991 Siegal et al., 1992), (c) some models of AICD (Strasser et al., 1991 Siegal et al., 1992) and (d) withdrawal of growth factor from IL-3-and IL-4-or GM-CSF-dependent haematopoietic cell lines (Vaux et al., 1988 Nunez et al., 1990). 

Nikonova, L.V., Nelipovich, P.A. and Umansky, S.R. (1982) The involvement of nuclear nucleases in rat thymocyte DNA degradation after gamma-irradiation. Biochim. Bio-phys. Acta 699 281-289. 

The first indication that in some systems B cell defects may explain nonresponder status was obtained by Shearer, Mozes and Sela in studies of the transfer of limited number of bone marrow cells and thymocj es to irradiated recipients to reconstitute their responses. Using this approach, they found that the defect in the response of SJL mice to (Phe,G)-A- -L could be attributed to both thymocytes and bone marrow cells, whereas with the antigen (Phe,G)-Pro- -L, the defect in SJL and DBA/1 mice was located only in bone marrow cells. ... 

HT medium add 1 ml of HT to 100 ml of DMEM containing 10% PCS Feeder cells for fosion coltures and cloning of hybridomas (essential for fusions using rat myelomas) rat fibroblast cell lines derived from the xiphistemae of various strains are suspended in DMEM and irradiated with about 30 Gy (3000 rad) of X- or gamma rays, then frozen in 95% FCS, 5% DMSO, and stored in liquid nitrogen as aliquots of 5 x 10 cells. Use one aliquot for each 200 ml of HAT or HT medium. Alternatively, use thymocytes from spleen donors. 

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