A better cell line for making hybridomas secreting specific antibodies

Shulman, Marc J.; Wilde, C. Deborah; Köhler, Georges

doi:10.1038/276269a0

Cited by 1,562 publications

(525 citation statements)

References 8 publications

Supporting

Mentioning

516

Contrasting

Unclassified

Order By: Relevance

“…The difference between these values can largely be explained by the difference in the DNA contents of the two cell populations. Sp2 cells are mouse splenocyte/myeloma hybrids with about 73 chromosomes (35), whereas human lymphocytes contain only 46 chromosomes. In addition, the Sp2 cells are from exponential culture with a DNA content approximating that of mid-S phase (PI-DNA fluorescence values were averaged over the whole population), whereas peripheral blood lymphocytes are naturally quiescent populations (26) in GO/G1.…”

Section: Discussion Pi Binding Sitesmentioning

confidence: 99%

“…The murine myeloma cell line Sp2/0-Ag14 (referred to here as Sp2) (35) was cultured and propagated as previously described (46) in RPMI 1640 (Biochrom, Berlin, Federal Republic of Germany) complete growth medium supplemented with 10% heat-inactivated fetal calf serum at 37°C in a humidified atmosphere enriched with 5% CO,. When they were harvested, the cultures were in exponential growth and asynchronous.…”

Section: Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

et al. 1995

View full text Add to dashboard Cite

Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmolkell) before electropulsation (0.5-3.0 kV/cm, 40 ps) in medium containing 25-50 pg/ml PI at 21-23°C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form >95% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic "pulse medium," the membranes resealed after electropulsing with a time constant (73 of about 2 min. In 150 mOsm medium, resealing was faster (typically T~ -0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius de-10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (C,), was taken into account. Evaluation of the C, values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. Key terms: Plasma membrane, electric breakdown, cytoplasm, fluorescent staining, propidium iodide, dye uptake, electrorotation, carboxyfluorescein, flow cytometry, cell viability Electropermeabilization allows the introduction of foreign substances (e.g., drugs, hormones, proteins, plasmids) into living cells (for reviews, see 27,44,45). This technique has been reported to be the most reliable for studying the effects of normally impermeant molecules on the regulation of cell metabolism. Other permeabilization methods (e.g., the use of bacterial toxins, detergents, hypertonic treatment) can markedly affect cell viability and cause undesirable leakage of the cytoplasm or extensive cell lysis ( 2 5 3 0 ) .Plasma membrane electropermeabilization within statistically representative cell samples can be assessed by measuring membrane crossing of tracer molecules in a flow cytometer. Extremely heterogeneous responses to electropulsing have been found in rather homogeneous (origin, size, morphology) cell samples despite the use of uniform electric fields (parallel plate electrodes) for several cell types (6,9,10,24). Flow cytometry of human red blood cells after electropulsation in the presence of FITCdextran (70 kDa) gave rise to at least three distinct subpopulations: ghosts with negligible upta...

show abstract

Section: Discussion Pi Binding Sitesmentioning

confidence: 99%

Section: Cellsmentioning

confidence: 99%

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

et al. 1995

View full text Add to dashboard Cite

show abstract

“…Two monoclonal antibodies (MAb) against TIMP-1, 33 NM4 (clone rTIX6A, NeoMarkers, Fremont, CA) and CalB2 (clone 147-6D11, CalBiohem, Oncogene Res. Products, Cambridge, MA), and a MAb against trinitrophenyl (TNP) 34 were all incubated at 1.0 g/ml (all 3 MAbs are IgG1). CalB2 MAb recognizes both free TIMP-1 and TIMP-1 in complex with MMPs.…”

Section: Immunoperoxidase Stainingmentioning

confidence: 99%

Localization of tissue inhibitor of metalloproteinases 1 (TIMP‐1) in human colorectal adenoma and adenocarcinoma

Holten-Andersen

Hansen

Brünner³

et al. 2004

Intl Journal of Cancer

View full text Add to dashboard Cite

Tissue inhibitor of matrix metalloproteases 1 (TIMP-1) inhibits the proteolytic activity of matrix metalloproteases and hereby prevents cancer invasion. However, TIMP-1 also possesses other functions such as inhibition of apoptosis, induction of malignant transformation and stimulation of cell-growth. We have previously demonstrated that TIMP-1 is elevated in blood from colorectal cancer patients and that high TIMP-1 levels predict poor prognosis. To clarify the role of TIMP-1 in colorectal tumorigenesis, the expression pattern of TIMP-1 in benign and malignant colorectal tumors was studied. In all of 24 cases of colorectal adenocarcinoma TIMP-1 mRNA was detected by in situ hybridization. In all cases TIMP-1 expression was found in fibroblast-like cells located at the invasive front but was seen only sporadically in normal mucosa. No TIMP-1 mRNA was seen in any of the cases in benign or malignant epithelial cells, in vascular cells or smooth muscle cells. Comparison of sections processed for TIMP-1 in situ hybridization with sections immunohistochemically stained with antibodies against TIMP-1 showed good correlation between TIMP-1 mRNA and immunoreactivity. Combining TIMP-1 in situ hybridization with immunohistochemical staining for ␣-smooth muscle actin or CD68 showed TIMP-1 mRNA in myofibroblasts but not in macrophages. TIMP-1 mRNA was detected in 2 of 7 adenomatous polyps in the adenoma area: in both cases associated with focal stromal inflammation at the epithelial-stromal interface. In conclusion, TIMP-1 expression is a rare event in benign human colon tissue but is highly expressed by myofibroblasts in association with invading colon cancer cells.

show abstract

“…A final boost was an intraperitoneal injection of the immunoglobulin fusion proteins without adjuvant. Spleen cells were fused with Sp2/0‐Ag14 mouse myeloma cells28 according to standard procedures 29…”

Section: Methodsmentioning

confidence: 99%

Functional annotation of the T‐cell immunoglobulin mucin family in birds

Vervelde

et al. 2016

Immunology

View full text Add to dashboard Cite

SummaryT‐cell immunoglobulin and mucin (TIM) family molecules are cell membrane proteins, preferentially expressed on various immune cells and implicated in recognition and clearance of apoptotic cells. Little is known of their function outside human and mouse, and nothing outside mammals. We identified only two TIM genes (chTIM) in the chicken genome, putative orthologues of mammalian TIM1 and TIM4, and cloned the respective cDNAs. Like mammalian TIM1, chTIM1 expression was restricted to lymphoid tissues and immune cells. The gene chTIM4 encodes at least five splice variants with distinct expression profiles that also varied between strains of chicken. Expression of chTIM4 was detected in myeloid antigen‐presenting cells, and in γδ T cells, whereas mammalian TIM4 is not expressed in T cells. Like the mammalian proteins, chTIM1 and chTIM4 fusion proteins bind to phosphatidylserine, and are thereby implicated in recognition of apoptotic cells. The chTIM4–immunoglobulin fusion protein also had co‐stimulatory activity on chicken T cells, suggesting a function in antigen presentation.

show abstract

A better cell line for making hybridomas secreting specific antibodies

Cited by 1,562 publications

References 8 publications

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

Localization of tissue inhibitor of metalloproteinases 1 (TIMP‐1) in human colorectal adenoma and adenocarcinoma

Functional annotation of the T‐cell immunoglobulin mucin family in birds

Contact Info

Product

Resources

About