Lymphocytes labelling

Figure 13. Microscope photo of human lymphocytes labeled by means of the direct method (the lymphocytes were not sensitized prior to labeling) with 0.7 It. microspheres goat antihuman antibody conjugates...

Garson JA, Beverley PCL, Coackhan HB, Harper El (1982) Monoclonal antibodies directed against human T lymphocytes label Purkinje neurones of many species. Nature, 298, 375-377. 

Fig. 3. The effect of adding 2 mM 3AB at various times after 1.5 Gy of X-rays on die induction of chromatid breaks in human lymphocytes labeled with 0.1 pCiAnl pH]dThd for 6 hr 24 hr after mitogen stimulation. The vertical bars indicate standard error of the mean. At each time point the number of induced aberrations was calculated by subtracting the number of background breaks and the number of [%QdThd-induced chromatid breaks from the total number of aberrations observed. For comparison, the number of X-ray-induced aberrations in cells not exposed to pHJdThd is indicated by the dashed horizontal line. Reprinted with permission from Wiencke etal. (10).

Loeffler, D. Ratner, S. In vivo localization of lymphocytes labeled with low concentrations of Hoechst 33342. J. Immunol. Methods 1989, II9, 95-101. 

Figure 18. (a) Immunocytochemical labeling of B-lymphocytes in muscle from a patient with juvenile dermatomyositis. (b) Immunocytochemical labeling of T8 lymphocytes showing that the infiltrate contains very few cells of this type (serial section). 

Figure 20. Immunocytochemical labeling of T8 lymphocytes showing some cells in close contact with muscle fiber plasma membranes (arrowheads).

LG j8-Lact< obulin LGL Large granular lymphocyte LH Luteinizing hormone LHRH Luteinizing hormonereleasing hormone LI Labelling index LIS Lateral intercellular spaces LMP Low molecular mass polypeptide... 

Reported applications of BSOCOES include studying the polypeptide antigens on lymphocyte cell surfaces (Zarling et al., 1980), crosslinking labeled (3-endorphin to its opioid receptors (Howard et al., 1985), and isolation and characterization of calcitonin receptors in rat kidney (Bouizar et al., 1986). 

Lymphocytes, the effector cells of the acquired immune system, include morphologically indistinguishable T and B cells, the former divided into CD4+ T helper cells and CD8+ cytotoxic T cells. Since the functions of those cell subsets differ so drastically, it became important to develop tools to distinguish them from each other. Efforts to identify cell subsets according to their expression of different surface antigens have been successful, including various Cluster of Determination (CD) markers (Table 23.1). In addition, cross-reactive monoclonal antibodies, and subsequently developed species-specific polyclonal and monoclonal antibodies towards the major histocompatibility complex (MHC) have been used to label cells in circulation and in tissue sections (Table 23.1). 

Nickel sulphate in peripheral blood T lymphocytes gave a marked increase of 32P label into nonhistone proteins [ 109], especially in the 30-40 kDa region. It was postulated that the increase in nuclear protein phosphorylation probably reflected an activation of the lymphocytes. 

No difference in the labelling of lymphocytes with 63Ni could be found in lymphocytes from nickel-allergic and control subjects [307, 308], approximately 20% of the lymphocytes being labelled. A difference was found regard-... 

Total RNA is isolated from the lymphocytes according to standard procedures and used as a template for radioactive labeled cDNA synthesis. The purified cDNA is used as probe for cDNA expression arrays. The advantages of this method as compared to other array systems are as follows (1) Radioactive-labeled probes are more sensitive than fluorescent-labeled probes and therefore need less sample RNA. (2) The primers used in the cDNA synthesis match the genes represented on the array. (3) The primer sequences are longer compared to other array systems, which increases the hybridization fidelity of RNA to the matching correct set of genes and therefore reduces mismatch reactions. 

Geoghegan, W. D., Scillian, J. J., and Ackerman, G. A. (1978) The detection of human B lymphocytes by both light and electron microscopy utilizing colloidal gold labeled anti-immunoglobulin. Immunol. Commun. 7, 1-12. 

Mammalian cells in culture are exposed to the test substance. Established cell lines are treated both with and without metabolic activation. Cells are incubated for an appropriate length of time, then rinsed, fixed, and dried. Slides are developed, stained, and exposed silver grains are counted. The endpoint of UDS is measured by determining the uptake of labeled nucleosides in cells that are not undergoing scheduled (S-phase) DNA synthesis. The most widely used technique is the determination of the uptake of H-TdR by autoradiography. Primary cultures (e.g., rat hepatocytes), human lymphocytes, or established cell lines (e.g., human diploid fibroblasts) may be used in the assay. Multiple concentrations of the test substance over a range adequate to define the response, should be used. 

Fig. 9. Flow analysis of apoptotic human peripheral blood lymphocytes using direct TUNEL assay. Human peripheral blood lymphocytes (1 x 10 ) treated (A) without or (B) with dexamethasone (0.1 ijlM) for 16 h were transferred to a 15-ml tube. Paraformaldehyde (2%) was added to cells with shaking and incubated for 10-15 min in ice with occasional shaking. Cells were washed with PBS with 1% BSA and 3-4 ml cold acetone was then added to cells. After 2-3 min incubation on ice with occasional shaking, cells were washed twice and TUNEL reaction mixture including enzyme TdT and fluorescein-labeled anti-dUTP antibody was added to cells (H). For the negative control group, only label solution without TdT was added to cells ( ). Cells without any addition of reaction or label solution were used for assessment of the autofluorescence ( ). The cell mixture was incubated 1 h at 37°C in the dark. The result of the apoptosis after flow analysis was expressed as a histogram using software CellQuest (Becton Dickinson). In Fig. 9A Ml (nonapoptotic cells), 86% M2 (apoptotic cells), 14%. In Fig. 9B Ml (nonapoptotic cells), 78% M2 (apoptotic cells), 22% (our unpublished data).

Figure 10.8. Outline of the production strategy of CEA-SCAN. The antibody-producing hybridoma cell line was originally obtained by standard methods of hybridoma generation. Spleen-derived murine B-lymphocytes were fused with murine myeloma calls. The resulting stable hybridomas were screened for the prodnction of anti-CEA monoclonals. The clone chosen produces an IgG anti-CEA antibody. Note that the finished product outlined above is not radio-labelled. The freeze-dried antibody preparation (which has a shelf-life of 2 years at 2-8°C) is reconstituted immediately prior to its medical use. The reconstituting solution contains Tc, and is formulated to facilitate direct conjugation of the radio-label to the antibody fragment...

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