[27] Flow cytometry: Rapid isolation and analysis of single cells

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.1.408-412.2003 ◽

2003 ◽

Vol 47 (1) ◽

pp. 408-412 ◽

Cited By ~ 21

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.

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

Journal of Microbiological Methods ◽

10.1016/j.mimet.2019.105745 ◽

2019 ◽

Vol 166 ◽

pp. 105745 ◽

Cited By ~ 1

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

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

Water Science & Technology ◽

10.2166/wst.1995.0181 ◽

1995 ◽

Vol 32 (4) ◽

pp. 177-182 ◽

Cited By ~ 12

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.

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Heterogeneity in the Rate of Benzo[a]pyrene Metabolism in Single Cells: Quantitation Using Flow Cytometry

Molecular and Cellular Biology ◽

10.1128/mcb.2.6.625 ◽

1982 ◽

Vol 2 (6) ◽

pp. 625-632 ◽

Cited By ~ 17

Author(s):

Arthur G. Miller ◽

James P. Whitlock

Keyword(s):

Flow Cytometry ◽

Enzyme Activity ◽

Cell Line ◽

Single Cells ◽

Hepatoma Cells ◽

Microsomal Enzyme ◽

Clonal Population ◽

Enzyme Inducer ◽

Mouse Hepatoma ◽

Low Activity

We describe a method for quantitating heterogeneity in the rate of benzo[a]pyrene metabolism in single cells by using flow cytometry. We have used the technique to study the response of Hepa-1c1c7 mouse hepatoma cells to the microsomal enzyme inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cells responded in a relatively homogeneous fashion at different times of induction with a maximally inducing concentration of the inducer. However, the induction response could be heterogeneous at a submaximal inducer concentration. We found even higher heterogeneity of enzyme activity among low-activity variants derived from the Hepa-1c1c7 cell line. When cells of either high or low activity were isolated from such a clonal population, propagated, and reanalyzed, they displayed average enzyme activity and heterogeneity identical to the parental cells; therefore, the heterogeneity represents transient, nonheritable differences between cells within the population.

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Comparison of HPV E6,E7 mRNA/PD-L1 assay using flow cytometry and p16 IHC expression in oral cancer samples.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.114 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 114-114 ◽

Cited By ~ 1

Author(s):

Yvette Carrasco ◽

Amanda Chargin ◽

Haitham Mirghani ◽

Bruce Kendrick Patterson

Keyword(s):

Flow Cytometry ◽

Squamous Cell Carcinoma ◽

Oral Cancer ◽

Cell Carcinoma ◽

Head And Neck ◽

Squamous Cell ◽

Single Cells ◽

Hpv Infection ◽

Neck Squamous Cell Carcinoma ◽

Hpv E6

114 Background: Research into causality of head and neck squamous cell carcinoma (HNSCC) has found a link to HPV infections affiliated with better survival than tobacco associated HNSCC. Currently, p16 immunohistochemistry is used as a predictive biomarker for HPV infection in HNSCC. We were interested in looking at an additional biomarker, HPV E6,E7 mRNA overexpression by flow cytometry, to see if it correlated with p16 status. Each test looks at a different marker of HPV infection, p16 as a surrogate of E7 activity, and E6,E7 mRNA overexpression as a marker of transcriptionally active and integrated virus. Currently p16 positive samples are confirmed with an ISH assay. Additionally, we looked at the PD-L1 expression in these tumors to see if it correlated with HPV mRNA overexpression. Advances in immuno-oncology have brought immunotherapy to the forefront of cancer treatment including HNSCC. Methods: Swabs were collected from Institut Gustave Roussy patients with lesions of the oral pharynx. Swabs were placed into a vial with a proprietary fixation solution and shipped overnight for processing. Upon receipt, samples were passed through a 35 uM filter to remove aggregates. Cells underwent in situ hybridization with E6, E7 mRNA probes (HPV OncoTect), were labeled with PD-L1 Ab, and then stained with a cell cycle dye identify single nucleated cells prior to analysis on the flow cytometer. FFPE biopsy tissue of the lesion was tested with p16 IHC. Positive samples were confirmed by ISH. Results: We analyzed samples from 27 patients with oral cancer with the combined E6, E7 mRNA/PD-L1 assay by flow cytometry and p16/ISH. Concordance between HPV E6,E7 mRNA positive results and p16 positive confirmed by ISH was 74%. Interestingly, PD-L1 expression was seen only in samples without HPV infection (according to HPV E6,E7 mRNA flow result). Samples are still being accrued and updated data will be presented at the meeting. Conclusions: Here we report a novel assay to quantify both HPV E6, E7 mRNA and PD-L1 simultaneously in single cells from head and neck squamous cell carcinoma. In addition, the ability to characterize both E6,E7 mRNA expression and PD-L1 in one test can provide clinicians with insight into treatment options.

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