Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells

Plovins, A; Álvarez, Alberto; Ibáñez, María del Mar Soria; Molina, Marı́a; Nombela, C

doi:10.1128/aem.60.12.4638-4641.1994

Cited by 71 publications

(30 citation statements)

References 12 publications

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“…Approximately 12,000 cells were analyzed in each sample. This cell number is twice the value reported by other researchers using the same (8) or similar substrates (23, 24)…”

Section: Methodssupporting

confidence: 60%

Population Dynamics of a Continuous Fermentation of Recombinant Saccharomyces cerevisiae Using Flow Cytometry

Chau¹,

Guillán

Roca³

et al. 2001

Biotechnol. Prog.

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The plasmid instability of genetically modified microorganisms during prolonged bioreactor operations is one of the major problems to be overcome in the production of recombinant proteins. The use of flow cytometry to monitor a fermentation process with recombinant cells in a CSTR is reported here. This technique has been applied to determine the fraction of plasmid-bearing cells (P+) of a recombinant Saccharomyces cerevisiae strain harboring the EXG1 gene in a continuous stirred tank bioreactor with a working volume of 2 L. The different levels in the expression of the EXG1 gene, which encodes the enzyme exo-beta-glucanase, were used to determine the P+ fraction. Other parameters such as viability, cellular protein, cell size and structure were also monitored using flow cytometry. This technique has two main advantages over the conventional method of determining the P+ fraction (plating in selective and non-selective solid media): (a) it takes a very short period of time to obtain a measurement that provides multiple parametric information; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample. A continuous operation (432 h) with the recombinant strain in a CSTR was carried out to test the application of this technique. Measurements of cellular exo-beta-glucanase activity and cellular protein content closely correlates to the measured fraction of plasmid-containing cells in the population. Moreover, the standard deviation of the fraction of P+ cells determined using this technique was very low (about 2%). Recombinant protein production also increased the size of the yeast cells, whereas the recombinant cells also had a more complex internal structure than the non-recombinant host strain.

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“…Approximately 12,000 cells were analyzed in each sample. This cell number is twice the value reported by other researchers using the same (8) or similar substrates (23, 24)…”

Section: Methodssupporting

confidence: 60%

Population Dynamics of a Continuous Fermentation of Recombinant Saccharomyces cerevisiae Using Flow Cytometry

Chau¹,

Guillán

Roca³

et al. 2001

Biotechnol. Prog.

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“…), Ministerio de Ciencia y Tecnología (to M.B. ), Ministerio FDG (Molecular Probes) protocol (Plovins et al, 1994) and sorted (MoFlo de Sanidad y Consumo and Comunidad Autó noma de Madrid (to C.G. ), and system, DakoCytomation).…”

Section: Western Blot Analysis Generation Of Knockin Miceunclassified

Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context

et al. 2003

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We have targeted a K-ras allele in mouse embryonic stem (ES) cells to express a K-Ras(V12) oncoprotein along with a marker protein (beta-geo) from a single bicistronic transcript. Expression of this oncogenic allele requires removal of a knocked in STOP transcriptional cassette by Cre recombinase. Primary mouse embryonic fibroblasts expressing this K-ras(V12) allele do not undergo proliferative senescence and proliferate as immortal cells. In mice, expression of K-ras(V12) throughout the body fails to induce unscheduled proliferation or other growth abnormalities for up to eight months. Only a percentage of K-ras(V12)-expressing lung bronchiolo-alveolar cells undergo malignant transformation leading to the formation of multiple adenomas and adenocarcinomas. These results indicate that neoplastic growth induced by an endogenous K-ras oncogene depends upon cellular context.

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“…The expression of bgal was measured using Fluorescein di-b-D-galactopyranoside (FDG). The protocol described below is a modification to previously published work (Plovins et al, 1994;Chappell & Jacob, 2006).…”

Section: Mouse Experimentsmentioning

confidence: 99%

The GTP ase Rab39a promotes phagosome maturation into MHC ‐I antigen‐presenting compartments

2019

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For CD8 T lymphocytes to mount responses to cancer and virallyinfected cells, dendritic cells must capture antigens present in tissues and display them as peptides bound to MHC-I molecules. This is most often accomplished through a pathway called antigen cross-presentation (XPT). Here, we report that the vesicular trafficking protein Rab39a is needed for optimal cross-presentation by dendritic cells in vitro and cross-priming of CD8 T cells in vivo. Without Rab39a, MHC-I presentation of intraphagosomal peptides is inhibited, indicating that Rab39a converts phagosomes into peptide-loading compartments. In this process, Rab39a promotes the delivery of MHC-I molecules from the endoplasmic reticulum (ER) to phagosomes, and increases the levels of peptide-empty MHC-I conformers that can be loaded with peptide in this compartment. Rab39a also increases the levels of Sec22b and NOX2, previously recognized to participate in cross-presentation, on phagosomes, thereby filling in a missing link into how phagosomes mature into cross-presenting vesicles.

show abstract

Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells

Cited by 71 publications

References 12 publications

Population Dynamics of a Continuous Fermentation of Recombinant Saccharomyces cerevisiae Using Flow Cytometry

Population Dynamics of a Continuous Fermentation of Recombinant Saccharomyces cerevisiae Using Flow Cytometry

Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context

The GTP ase Rab39a promotes phagosome maturation into MHC ‐I antigen‐presenting compartments

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