Monoclonal antibodies specific for sickle cell hemoglobin.

Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility.

Kallikrein-like activity in salivary glands using a new tripeptide substrate, including preliminary secretory studies and observations on mast cells.

The tripeptide substrate D-val-leu-arg-4-methoxy-2-naphthylamine gave a precise localization of reaction product in cryostat sections of aldehyde-fixed salivary glands from a number of species, with Fast Blue B as the capture reagent. In submandibular glands, there was strong staining of the granules in granular tubules of rats and hamsters and somewhat less in mice. Submandibular striated ducts showed variable periluminal staining in a finer granular form; it was abundant in guinea-pigs, strong in cats but somewhat less pronounced in dogs. Parotid glands contained less reactivity with none detectable in hamsters and guinea-pigs. In the rabbit, neither gland showed any reaction. Mast cells were densely stained in glands from cats and dogs; they were less reactive in rats and unstained in the other species. The closely related 7-amino-4-trifluoromethylcoumarin derivative of the tripeptide has been found highly satisfactory for assessing activity in submandibular saliva from cats. Preliminary functional studies indicate that an extensive rapid secretion of enzyme occurs into saliva on sympathetic stimulation, with a corresponding depletion of reactive material from the striated ducts in tissue sections. Far less mobilization of enzyme occurs into saliva on parasympathetic stimulation with no obvious change in the histochemical reaction of striated ducts. The possible significance of these findings in cats is discussed. Extensive qualitative and quantitative studies are required to evaluate enzyme and substrate specificities in each species. Nevertheless, derivatives of D-val-leu-arg offer great promise for the functional testing of kallikrein-like reactivity both by histochemical means on cells and biochemically in their secretions.

Application of flow cytometry and cell sorting to megakaryocytopoiesis.

We have employed flow cytometry (FCM) and cell sorting to quantitate and study megakaryocytes in mouse and rat femoral marrow following their 20- to 30-fold concentration by centrifugal elutriation (CE). This enrichment of megakaryocytes permitted the first determination of their DNA-related fluorescence by FCM analysis following DNA staining. Fluorescence distributions of CE-enriched cell fractions following supravital staining with Hoechst 33342 were similar to those following chromomycin A3 staining of ethanol-fixed cells. Microscopic examination of cells sorted onto glass slides on the basis of their DNA-related fluorescence following supravital staining together with specific acetylcholinesterase staining for megakaryocytes indicated that megakaryocytes generally increased in cell size with increasing DNA content. This technologic application represents a significant advance in the study of megakaryocytopoiesis, since the kinetics of either the normal or perturbed population can now be studied rapidly and quantitatively.

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.