Trypsinization Technique Research Articles

Novel quantitative proteomic analysis of influenza infected dendritic cells reveals a previously unappreciated immune modulatory protein and infection induced differential expression of DC proteins, with unexpectedly high membrane protein dynamics. (110.24)

Abstract Airway dendritic cells (DC) initiate the immune response (IR) to respiratory pathogens (e.g.influenza). DC respond by releasing cytokines and presenting antigen to T cells, which activate on encountering the MCHI/peptide complex. This DC-T cell interaction dictates the IR to a given pathogen. Given that antigen presentation couples the innate and adaptive immune responses, a better understanding of these processes is vital to basic immune research and to therapeutic and vaccine development. To elucidate the underlying mechanisms of these processes we elected to identify DC proteins and quantify their changes in abundance using 18O labeling and LTQ-Orbit-trap-MS. Presuming the import of membrane protein dynamics on DCs, we developed a high pressure trypsinization technique to denature and digest insoluble proteins. We identified >1500 DC proteins, 20% of which change in abundance (i.e. ≥ 2 fold) early in infection. Unexpectedly this method detected infection induced changes in membrane proteins exceeding soluble protein dynamics. Analysis of these preliminary data indicates major shifts in DC metabolic and antigen processing and presentation pathways occur as a result of infection. Network analysis resulted in the identification of two critical modulator proteins; one of these was NFκB, a well known modulator of the immune response and another protein previously unidentified as an IR modulator. Together these data revealed new and important aspects of the immune response.

Expression of innate immune proteins by human endometriosis cell line MD-E

OBJECTIVE: To characterize the innate immune proteins expressed by human endometriosis cell line MD-E and begin to determine modulation of expression by hormones.DESIGN: This is an in vitro descriptive study.MATERIALS AND METHODS: Human endometriosis cell line MD-E maintained in RPMI 1640 10% heat inactivated fetal calf serum in a CO2 incubator were grown to confluence and split using standard trypsinization technique. In the hormone modulation studies, cells were washed with culture medium by centrifugation, suspended in fresh medium, and plated in 12 well plates. When near confluent the medium was replaced with fresh medium with/without hormone (25 ng/ml cetrotide) for 24 hours. Cells were then harvested by trypsinization and stained for LL-37 and aromatase. For IHC the trypsinized cells were washed with PBS, spotted on positively charged slides, air dried and fixed for 10 min in cold 4° acetone. The fixed cells were stained using mouse monoclonal or affinity purified rabbit polyclonal antibodies and Dako Mouse or Rabbit Envision Plus Staining kits according to their protocol. For negative controls, rabbit or mouse DAKO control reagents were subsituted for primary antibodies. All slides were processed and evaluated using Leitz microscope.Table 1Expression of Innate Immune Factors and Aromatase by MD-ELL-37AromataseHAD1HAD5HAD6HBD2cryopyrinControl++++++++++++++-- Open table in a new tab Glucocorticoid, progesterone, and estrogen receptors were positive. Aromatase was stongly expressed. There was no change in expression of LL-37 or aromatase by cetrotide.CONCLUSIONS: Endometrosis cells can express multiple innate immune proteins, which may play a role in the pathophysiology of endometriosis via influencing the inflammatory process. MD-E strongly expressed LL-37, which has been associated with ovarian cancer. We plan to continue our studies on the potential effects of steroid signaling (estrogen, progesterone, dexamethasone and letrozole) on these innate immune factors. OBJECTIVE: To characterize the innate immune proteins expressed by human endometriosis cell line MD-E and begin to determine modulation of expression by hormones. DESIGN: This is an in vitro descriptive study. MATERIALS AND METHODS: Human endometriosis cell line MD-E maintained in RPMI 1640 10% heat inactivated fetal calf serum in a CO2 incubator were grown to confluence and split using standard trypsinization technique. In the hormone modulation studies, cells were washed with culture medium by centrifugation, suspended in fresh medium, and plated in 12 well plates. When near confluent the medium was replaced with fresh medium with/without hormone (25 ng/ml cetrotide) for 24 hours. Cells were then harvested by trypsinization and stained for LL-37 and aromatase. For IHC the trypsinized cells were washed with PBS, spotted on positively charged slides, air dried and fixed for 10 min in cold 4° acetone. The fixed cells were stained using mouse monoclonal or affinity purified rabbit polyclonal antibodies and Dako Mouse or Rabbit Envision Plus Staining kits according to their protocol. For negative controls, rabbit or mouse DAKO control reagents were subsituted for primary antibodies. All slides were processed and evaluated using Leitz microscope. Glucocorticoid, progesterone, and estrogen receptors were positive. Aromatase was stongly expressed. There was no change in expression of LL-37 or aromatase by cetrotide. CONCLUSIONS: Endometrosis cells can express multiple innate immune proteins, which may play a role in the pathophysiology of endometriosis via influencing the inflammatory process. MD-E strongly expressed LL-37, which has been associated with ovarian cancer. We plan to continue our studies on the potential effects of steroid signaling (estrogen, progesterone, dexamethasone and letrozole) on these innate immune factors.

Insulin internalization into monocytes is decreased in patients with type II diabetes mellitus.

We studied the internalization of [125I]insulin into circulating human monocytes, a cell type widely used for insulin binding studies. The internalization of [125I]insulin was assessed by both an acid extraction technique, which removes surface-bound insulin but not intracellular insulin, and by a trypsinization technique, which removes cell surface-bound hormone. After 5 h of incubation at 22 C, over 40% of the total cell-associated [125I]insulin was internalized into monocytes of normal subjects. This internalization was temperature dependent; the fraction of internalized hormone was progressively decreased when the incubation temperature was reduced from 37 to 4 C. Treatment of monocytes with increasing concentrations of 2,4-dinitrophenol also decreased [125I]insulin internalization, whereas dansylcadaverine, an inhibitor of transglutaminase, had no effect. Analysis by gel filtration of the internalized labeled hormone after 4 h of incubation at 22 C indicated that 50-60% of the label was degraded insulin, but detectable intact insulin was still present. Internalization of insulin was then studied in monocytes from eight obese patients (161% of ideal body weight) with type II diabetes mellitus. After 4 h of incubation at 22 C, the specific total monocyte-associated [125I]insulin was decreased compared to that in cells from 7 normal subjects [6.02 +/- 0.38% (+/- SE) vs. 3.91 +/- 0.31% of the total; P less than 0.001]. Moreover, the percentage of hormone that was internalized was also decreased from 41.4 +/- 1.2% of the total to 28.9 +/- 1.8% (P less than 0.001). In 20 nondiabetic obese subjects, specific cell-associated [125I]insulin was reduced to 3.9 +/- 0.3% (P less than 0.001). However, compared to that in normal subjects, the percentage of hormone that was internalized was not decreased (39.7 +/- 3.51% of the total). The present findings indicate that human circulating monocytes internalize [125I]insulin; this process is temperature and energy dependent; and monocytes from obese type II diabetic patients have a significantly decreased ability to internalize insulin. This decreased internalization may play a role in the cellular resistance to insulin that occurs in these patients.

Serial propagation of mammalian cells on microcarriers

For the large-scale operation of microcarrier culture to be successful, a technically feasible method for sequential inoculation is essential. Using human foreskin fibroblasts, FS-4, we have achieved this by detaching cells viably from microcarriers employing a selection pH trypsinization technique. Cells thus detached are able to reattach to microcarriers and grow normally after subsequent reinoculation into new cultures. However, after reinoculation cells attach to new microcarriers at a higher rate than to used microcarriers on which cells have previously grown. The effect of this differential cell attachment was analyzed and overcome by employing a low inoculum concentration. FS-4 cells could thus be serially propagated on microcarriers and subsequently used for beta-interferon production. This technique has also been applied to the cultivation of a monkey kidney cell line, Vero. We have also shown that Vero cells directly inoculated from a seed microcarrier culture could be used for virus production.

Acetylation of histones during spermatogenesis in the rat

Acetate was actively incorporated into rat testis histones when testis cells were prepared by the trypsinization technique in the presence of [ 3H]acetate. The acetylation was enhanced by 10 m m sodium butyrate. Although histones H3 and H4 were the only histones which incorporated high levels of acetate, the testis-specific histones TH2B and TH3 also appeared to incorporate acetate. This was shown by electrophoresis of the histones on polyacrylamide gels containing Triton X-100. Results, obtained from analysis of histones by two-dimensional gel electrophoresis, confirmed a recent report (P. K. Trostle-Weige, M. L. Meistrich, W. A. Brock, K. Nishioka, and J. W. Bremer, (1982) J. Biol. Chem. 257, 5560–5567) that TH2A was a testis-specific histone. The results also confirmed the H2A nature of a testis-enriched histone band, previously designated X2. When histones from populations of cells enriched in specific testis cell types, representing various stages of spermatogenesis, were examined, the patterns of acetylation varied dramatically. Very high levels of acetate were incorporated into multiacetylated species of histone H4 from a population of cells enriched in transition stage spermatids (steps 9–12) compared to the levels of acetate incorporated into H4 from round spermatids (steps 1–8) and earlier stages of spermatogenesis, where acetate was incorporated primarily into the monoacetylated species of H4. Thus, a striking correlation exists between the time of hyperacetylation of histone H4 and the time of removal of histones for their replacement by the basic spermatidal transition proteins designated TP, TP2, and TP4. Hyperacetylation of histone H4 may facilitate the removal of the entire histone complement during the protein transition. In any case, it must be an obligatory step in the dramatic process.

Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of Swiss 3T3 cells.

Improved culture conditions have been developed that will support clonal growth of Swiss mouse embryo 3T3 cells at concentrations of serum protein at low as 125 micrograms/ml. Survival of the cells under completely protein-free conditions also is enhanced greatly. The improvements that made these results possible include: (a) use of medium MCDB 402, which was developed specifically for Swiss 3T3 cells by adjusting the concentrations of all components of Dulbecco's modified Eagle's medium to optimum values for clonal growth with minimal serum protein and by adding other nutrients such as trace elements and "nonessential" amino acids that were not in the original formula; (b) use of culture surfaces that are coated with a positively charged polymer, poly-D-lysine; and (c) use of gentle low temperature trypsinization technique that minimizes cellular damage and the need to neutralize residual trypsin.

Radiobiological Studies of Cells in Multicellular Spheroids Using a Sequential Trypsinization Technique

The radiation response of V79 Chinese hamster cells grown as multicellular spheroids has been investigated by determining survival curves for treatment under a variety of different oxygen concentrations. Spheroids were irradiated under fully oxygenated conditions in air-equilibrated medium at 37/sup 0/C, in medium exposed to lower oxygen tension (5% O/sub 2/) for times varying from 1 hr to 3 days, or under anoxic conditions. For comparison with the spheroids, using identical treatment conditions, V79 cells were grown in suspension as a subconfluent monolayer attached to Sephadex (microcarrier) beads and irradiated under fully oxygenated or anoxic conditions. The radiation response of cells at different depths within the spheroid was investigated by using a sequential trypsinization technique developed to remove eight or nine shells of cells successively from the spheroid surface. When irradiation was given under fully oxygenated conditions the outer few cell layers were more sensitive than the inner cells, a finding which is not understood. As expected the inner cells in spheroids irradiated in air (at 37/sup 0/C) or in 5% O/sub 2/ are more resistant than the outer cells. For an acute exposure to 5% O/sub 2/ (1 hr) in the inner cells displayed full radiobiological hypoxia; however, formore » chronic exposures to low oxygen this was not the case. These results with the sequential trypsinization procedure suggest that the radiation response of cells in spheroids is more complex than anticipted.« less

Establishment and characterization of 5 human cell lines derived from a series of 50 primary intracranial tumors

A series of 50 human primary intracranial tumors were cultivated in vitro in an attempt to establish cell lines with the trypsinization technique. During the in vitro adaptation period, cultures were maintained at high cell density to avoid rapid over-growth by connective tissue. Five lines were established from 5 tumors including 4 malignant gliomas (SA4, SA44, SA45, SA101) and 1 meningeal sarcoma (SA39). These 5 lines were compared for the following investigations and criteria: light and electron microscopy, chromosomal patterns, saturation density, plating efficiency, doubling time, synthesis of collagen, S-100 and GFA proteins, response to d b-c AMP and tumorigenicity into nude mice. For one or more of the characteristics mentioned above each line was distinct from the others. None of the growth parameters that serve to define the abnormal state in vitro correlated consistently with cellular tumorigenicity in athymic nude mice. The nature of lines derived from gliomas remain uncertain and the collagen synthesis in vitro is not sufficient to affirm that these lines are mesenchymal rather than glial.

Behaviour of the rectal juvenile polyp in vitro.

The behaviour of the juvenile polyp in vitro and its cellular development from explants have been studied using cellular dispersion by the trypsinization technique. Only the fibroblastic cells have been found to grow. The significance of this finding suggests that the juvenile polyp is a hamartoma.

High-yield method for dispersing simian kidneys for cell cultures.

A technique for dispersion of animal tissue cells is described. The proposed technique is based on the concomitant use of trypsin and disodium ethylenediamine tetraacetate (EDTA). The use of the two dispersing agents (trypsin and disodium EDTA) markedly enhances cell yield as compared with the standard cell dispersion methods. Moreover, significant reduction in the amount of time required for complete tissue dispersal, presence of a very low number of nonviable cells, less cell clumping, and more uniform monolayer formation upon cultivation compare favorably with the results usually obtained with the standard trypsinization technique.

High-Yield Method for Dispersing Simian Kidneys for Cell Cultures

A technique for dispersion of animal tissue cells is described. The proposed technique is based on the concomitant use of trypsin and disodium ethylenediamine tetraacetate (EDTA). The use of the two dispersing agents (trypsin and disodium EDTA) markedly enhances cell yield as compared with the standard cell dispersion methods. Moreover, significant reduction in the amount of time required for complete tissue dispersal, presence of a very low number of nonviable cells, less cell clumping, and more uniform monolayer formation upon cultivation compare favorably with the results usually obtained with the standard trypsinization technique.

Homogeneous cultivation of animal cells for the production of virus and virus products.

AbstractHomogeneous technique facilitates the cultivation of large quantities of cells, reduces the risk of contamination by eliminating many manipulations, and makes practical the control of conditions such as pH and oxygen tension. Although most animal cells will not multiply in free suspension, certain cell lines have lost the requirement of being attached to a solid surface. These cells can be subcultured indefinitely but have some resemblance to cancer cells such as their abnormal karyotype. Certain cell linen developed from human embryonic tissue maintain their diploid character after repeated subculture and would seem to be ideal for the production of vaccines. However, strict regulations exist for viral products for human injection in that only cells taken from normal tissue and subcultured but once may be used.A microcarrier method in which cells adhere to DEAE‐Sephadex beads permits a suspension culture which may be termed quasihomogeneous. The attached cells may be retained by sedimentation or by screening as the medium is replaced. Cell debirs from the original tissue is difficult to remove from microcarrier cultures; modifications of the trypsinization technique have alleviated but not solved this problem.Conditions for virus replication can be less critical than those for cell growth in that oxygen tension seems to have little influence on virus production. In cases where rate of virus production increases with specific growth rate of cells, homogeneous culture would have a advantage in maintaining a high cell mogeneous culture would have a valuble advantage in maintaining a high cell growth rate for a longer time. Some virus infections destroy cells, but others cause little change in cellular mteabolism except that virus is continually produced. The latter type can be conducted with a microcarrier in continuous culture with a virus titer exceeding 107 plaque forming units per milliliter for over 50 days with Rubella‐infected BHK cells.

SOME CHARACTERISTICS OF THE SHEEP THYROID GLAND SYSTEM FOR INCORPORATING AMINO ACIDS INTO PROTEIN.

The ability of sheep thyroid glands to incorporate the 14C of a variety of amino acids uniformly labeled with 14C into protein was studied in: (a) slices and (b) dispersed cells obtained by the continuous-flow trypsinization technique and cultured as monolayers. In slices, the 14C of the protein formed from valine 14C was located predominantly in mitochondrial and microsomal fractions. Addition of glucose, pyruvate, succinate or a-ketoglutarate to the incubation medium did not enhance incorporation of leucine-14C into protein by sheep thyroid slices. Thyrotropic hormone, added in vitro, had no effect on incorporation of leucine-14C into protein or on the uptake of 14C of α-amino isobutyric acid-l-14C by thyroid slices. Analysis of N- and C-terminal groups of 14C-proteins isolated from thyroid slices or cells incubated with the 14C-amino acids indicated that the incorporation took place predominantly at the interior sites of the protein molecules. Carbon from aspartate was incorporated into proteins by thy...

STIMULATORY EFFECT OF THYROTROPIN ON SYNTHESIS OF THYROXINE BY ISOLATED THYROID CELLS.

Iodide transport and thyroxine synthesis were studied in isolated cells prepared by trypsinization of bovine thyroid glands. When the cells were incubated in media containing 0.2 unit of thyrotropin (TSH) per ml, incorporation of I into thyroxine (and iodotyrosines) was stimulated 2to 4-fold, while no enhancement of iodideconcentrating activity could be detected. (Endocrinology 74: 304, 1964) D cells, obtained from ovine thyroid tissue by trypsinization, have been shown to retain the capacity to concentrate iodide, and to incorporate I-labeled iodide into iodotyrosines and iodothyronines (1). In the present study, the effects of added thyrotropin on these functions of the isolated cell preparation were investigated. Materials and Methods Dispersed thyroid cells were prepared by means of the continuous-flow trypsinization technique described previously (1). The following modifications of the technique were used in the present study: (a) bovine thyroid tissue was used instead of ovine tissue, (b) Earle's balanced salt solution was used in place of Hank's solution to prepare the trypsinizing reagent, (c) the freshly collected cells were washed with a dilute DNase solution to eliminate sliminess so that homogeneous cell suspensions could be obtained. In each experiment 2-3 ml of a fresh thyroid cell preparation was suspended in 50 ml of Earle's solution containing 4XlO~ M Nal labeled with I. 5 ml aliquots of such cell suspensions, containing 0.2-0.3 ml of cells, were delivered into 25 ml Erlenmeyer flasks and incubated under 95 % O2-5 % CO2 with shaking (90 oscillations/min) at 37 C in a Dubnoff water bath. After incubation, the contents of the flasks were transferred to Shevky-Stafford type centrifuge tubes, and the cells were sedimented by centrifugation for 5 min at 50 Xg in an International centrifuge maintained at a temperature of 5 C. Packed cell volumes were read to the nearest 0.01 ml, and then the supernatant media were drawn off. Iodine-concentrating activity was expressed as C/M ratios, I-iodide per ml cells I-iodide per ml medium The formation of organically bound I was prevented by incubating in the presence of 0.002 M Tapazole, so that I assays performed on packed cell pellets and aliquots of incubating media provided a direct measure of I-iodide distribution. However, the C/M values presented below are not corrected for the volume of the media trapped in the packed cell pellets. Extracellular space determined with C-inulin and I-albumin amounted to 49-60% of the packed cell volumes; hence, the true intracellular I-iodide concentrations were probably about twice those indicated by the uncorrected C/M values. In the chromatographic studies, I-labeled cells were homogenized in 10 volumes of 0.03 M tris buffer solution (pH 7.5) which contained 0.5 % (w/v) of Pronase. After incubation for 6-9 hr at 37 C, 200 /J aliquots of the hydrolysates were chromatographed on Whatman No. 3 MM paper in collidine-3N NH40H (1). r e labeled components were located by radioautography and assayed by means of a well-type Nal crystal, scintillation detector. Received September 4, 1963. Aided by USPHS Grant AM-06439. 1 Worthington desoxyribonuclease I. 2 Purchased from Fisher Scientific Co. 3 Abbreviations: Tapazole for 1-methyl, 2mercaptoimidazole; TSH for thyroid-stimulating hormone; MIT for 3-iodotyrosine; DIT for 3,5-diiodotyrosine; T4 for thyroxine; and tris for tris (hydroxymethyl) amino methane. 4 Pronase, Streptomyces griseus protease, was obtained from California Corporation for Biochemical Research.