Cell shape and density are critical to the evaluation of neutrophil function and/or activation. Dimethyl sulfoxide-cryofixation-freeze-substitution processing (DCF) instantly preserves cell processes and ultrastructural elements with fewer artifacts than routine chemical fixation with glutaraldehyde and postfixation osmium tetroxide (GO). This study morphometrically examined density-separated neutrophils to assess differences in DCF and GO processing procedures and studied the effect of dimethyl sulfoxide followed by GO fixation (DGO) on morphology. Fifteen consecutive neutrophils were analyzed using computerized planimetry for differences in DCF v. GO treatments (n = 4) and DGO v. GO treatments (n = 4). Cryofixed and DGO-fixed cells were significantly rounder than GO cells which had a more irregular surface with membrane projections. The cell volume of GO cells was 27-30% smaller than in DCF or DGO processing, while the surface area was similar. The increased volume in DCF and DGO cells did not appear to be due to abnormal cell swelling, since membranes, nuclear envelope, and mitochondrial cristae were more intact than in GO cells. Preservation of mitochondria as well as endocytic caveolae with a subplasmalemmal coating was best in DCF samples, moderate in DGO, and poorest in GO. Morphometric data showed that the nuclear compartment was 22% smaller, while the cytoplasm (and its associated compartments) was 29% smaller in GO compared to DCF-processed neutrophils. This was consistent with the more dense cytoplasm in GO cells. Pretreatment of neutrophils with dimethyl sulfoxide (DMSO) resulted in volume preservation and improved the morphology of GO fixation. In summary, DCF appears to be an excellent method for preserving neutrophil membranes and cytoplasmic organelles (particularly mitochondria), and prevents a number of artifacts caused by routine GO fixation. Morphology can also be improved by using DMSO in conjunction with GO.
Cryofixation instantly preserves cell processes without chemical alteration and freeze-substitution (FS) preserves cell constituents. Investigation has shown that cell membranes and microfilaments appear better preserved in cryofixed-freeze-substituted (CFFS) specimens, including basophils. However, the cells have not been looked at morphometrically nor have neutrophils been looked at specifically.Peripheral blood was collected in heparin from four male donors by venipuncture. Blood was centrifuged at 500 x g for 45 min to separate the granulocytes using a one-step density gradient approach, and then washed in RPMI 1640. Cells were split and half were resuspended in RPMI with 10% DMSO, cryofixed using the Eiko RF-2 and stored in liquid N2 until FS processing in 4% OsO4 in acetone with embedment in Spurr’s resin. The other half were resuspended and fixed in 3% glutaraldehyde in 0.1M Cacodylate, pH 7.35 for 1 hr at 4-6°C. Cells were dehydrated, embedded in Spurr’s resin, counterstained with UALC and examined using a Zeiss EM109.
Myeloid cells are known to contain myeloperoxidase (MPO) and catalase. This study has used MPO and catalase replete and deficient myeloid cell lines to clarify the localization of these components using 3,3’-diaminobenzidine (DAB) ultrastructural cytochemistry. Conditions of DAB incubation can be modified to preferentially stain catalase (alkaline at pH 9.7) or MPO (neutral at pH 7.0-7.6), but crossreactivity persists, preventing the discrimination between catalase and peroxidase. Biochemical assays demonstrated both MPO and catalase in HL60 cells; similar amounts of catalase but no MPO activity in the A7 cell line; increased amounts of catalase but no MPO activity in the HP50 and HP100 cell lines; and neither MPO nor catalase in the KG1 cell line. Neutral DAB stained MPO (pH 7.4; [DAB] 5 or 20 mg/10 mL 0.05 M Tris; 30 min or 120 min; 24° or 37°C; 0.01% H2O2) in HL60 (Fig. 1), but not in A7. Alkaline DAB intensely stained catalase (pH 9.7; 20 mg/10 mL; 120 min; 37°C; 0.01% or 0.03%) in A7.
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