Fast intracellular pH determination in single cells by flow-cytometry

1981 ◽  
Vol 68 (5) ◽  
pp. 265-266 ◽  
Author(s):  
G. Valet ◽  
A. Raffael ◽  
L. Moroder ◽  
E. W�nsch ◽  
G. Ruhenstroth-Bauer
Keyword(s):  
Flow Cytometry ◽  
Intracellular Ph ◽  
Single Cells

scholarly journals Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0240769
Author(s):  
Prasanna Channathodiyil ◽  
Jonathan Houseley
Keyword(s):  
Flow Cytometry ◽  
Transcriptome Analysis ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cyclin B1 ◽  
Immunofluorescent Staining ◽  
Rna Seq ◽  
Simple Method ◽  
Staining Methods

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

scholarly journals Optogenetic tool to spatiotemporally manipulate intracellular pH within single cells

2020 ◽  
Author(s):  
Caitlin Donahue ◽  
Michael Siroky ◽  
Katharine White
Keyword(s):  
Intracellular Ph ◽  
Single Cells

Intracellular pH evolution during batch cultures using flow cytometry

1994 ◽  
pp. 351-353
Author(s):  
M. Cherlet ◽  
N. Petitpain ◽  
M. Dardenne ◽  
P. Franck ◽  
J.M. Engasser ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Intracellular Ph ◽  
Batch Cultures

scholarly journals Flow Cytometric Assessment of Susceptibilities of Streptococcus pyogenes to Erythromycin and Rokitamycin

2003 ◽  
Vol 47 (1) ◽  
pp. 408-412 ◽  
Author(s):  
Pier Carlo Braga ◽  
Cinzia Bovio ◽  
Maria Culici ◽  
Monica Dal Sasso
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Streptococcus Pyogenes ◽  
Single Cells ◽  
Membered Ring ◽  
Flow Cytometric ◽  
Live Bacteria ◽  
Erythromycin A ◽  
Set Up ◽  
Syto 9

ABSTRACT The effects of erythromycin (a 14-membered ring macrolide) and rokitamycin (a 16-membered ring macrolide) on the viability of the Streptococcus pyogenes M phenotype were studied by means of flow cytometry and fluorescence microscopy by using a combination of two fluorochromes (syto 9 and propidium iodide) that stains live bacteria green and dead bacteria red. In order to apply the flow cytometry, a bacterial sonication procedure was expressly set up to separate single cells from the long, intralaced S. pyogenes chains of up to 30 to 40 cells that have previously prevented the application of flow cytometry to this type of bacteria. The association of flow cytometry using an appropriate sonication procedure, together with a combination of fluorescent probes, offered the possibility of very quickly investigating the different microbiological effects of rokitamycin at 2 μg/ml, which was active on the S. pyogenes M phenotype, and of erythromycin at doses of up to 32 μg/ml, which was not.

scholarly journals Intracellular pH, esterase activity, and DNA measurements of human lung carcinomas by flow cytometry

Cytometry ◽  
1990 ◽  
Vol 11 (3) ◽  
pp. 341-348 ◽  
Author(s):  
F. Liewald ◽  
N. Demmel ◽  
R. Wirsching ◽  
H. Kahle ◽  
G. Valet
Keyword(s):  
Flow Cytometry ◽  
Intracellular Ph ◽  
Esterase Activity ◽  
Human Lung ◽  
Lung Carcinomas ◽  
Dna Measurements

scholarly journals Measurement of intracellular pH using flow cytometry with carboxy-SNARF-1

Cytometry ◽  
1993 ◽  
Vol 14 (8) ◽  
pp. 916-921 ◽  
Author(s):  
Eric D. Wieder ◽  
Haiying Hang ◽  
Michael H. Fox
Keyword(s):  
Flow Cytometry ◽  
Intracellular Ph

scholarly journals Estimating RNA numbers in single cells by RNA fluorescent tagging and flow cytometry

2019 ◽  
Vol 166 ◽  
pp. 105745 ◽  
Author(s):  
Mohamed N.M. Bahrudeen ◽  
Vatsala Chauhan ◽  
Cristina S.D. Palma ◽  
Samuel M.D. Oliveira ◽  
Vinodh K. Kandavalli ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cells ◽  
Fluorescent Tagging

Flow cytometry: a powerful tool in analysis of biomass distributions in phytoplankton

1995 ◽  
Vol 32 (4) ◽  
pp. 177-182 ◽  
Author(s):  
R. R. Jonker ◽  
J. T. Meulemans ◽  
G. B. J. Dubelaar ◽  
M. F. Wilkins ◽  
J. Ringelberg
Keyword(s):  
Flow Cytometry ◽  
Large Range ◽  
Single Cells ◽  
Cell Types ◽  
Spectrometric Analysis ◽  
Analysis Techniques ◽  
Field Samples ◽  
Phytoplankton Monitoring ◽  
Correlated Information ◽  
Taxonomic Groups

The large range in concentrations and cell-sizes of algal cells and colonies and the large variety of cell types are the main reasons for developing a dedicated cytometer for the analysis of phytoplankton. A European Community funded consortium has developed the EurOPA cytometer, which is easily transported and can be operated at sea. With the EurOPA, both small single cells and large colonies of cyanobacteria can be analyzed in one run. This provides correlated information on optical characteristics, pigments contents and taxonomy. The resulting distribution of (chlorophyll) biomass over taxonomic groups can be inter-calibrated with standard spectrometric analysis techniques. The EurOPA can be used successfully for analysis of field samples and phytoplankton cultures. It is well suited for phytoplankton monitoring and grazing studies.

scholarly journals Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

2009 ◽  
Vol 75 (13) ◽  
pp. 4550-4556 ◽  
Author(s):  
Vicky G. Kastbjerg ◽  
Dennis S. Nielsen ◽  
Nils Arneborg ◽  
Lone Gram
Keyword(s):  
Listeria Monocytogenes ◽  
Single Cell ◽  
Intracellular Ph ◽  
Bacterial Population ◽  
Single Cells ◽  
Viable Cell ◽  
Population Subdivision ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Counts

ABSTRACT Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pHi was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pHi 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.

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