If an adherent cell line is used, ensure that the cells are subconfluent. The following immunostaining protocol outlines the steps required for detecting surface or intracellular antigens in adherent cells by flow cytometry. The progenitor specificity of this assay is still dubious as most of the adherent cells mimic even angiogenic macrophages by expressing marker of endothelial and myeloid lineage. Quantification of bacterial invasion into adherent cells by. Such a metachromatic property allows a relatively simple identification of cells with a high or a low mmp by flow cytometry and has been used in different cell models by several groups. Flow cytometry protocol for intracellular targets using detergents the following flow cytometry protocol for staining intracellular molecules using detergents to permeabilize cell membranes has been developed and optimized by biotechne. Here we describe methods for both tissue culture cell lines and adherent tissue culture cell lines. Single cells must be suspended at a density of 105107 cells ml to keep the narrow bores of the flow cytometer and its tubing from clogging up. Phosphate buffered saline pbs is a common suspension buffer. The method is simple to perform, less time consuming, with no cell loss and makes highthroughput flow cytometry on adherent cells a reality.
Finally on many occasions i have wanted to see how flow cytometry results compare with what the cells actually look like so i often culture the cells on microscope slides or on chamber slides, stain and look. Cell preparation for flow cytometry thermo fisher scientific. To address this we developed a cold trypsinphosphorylationspecific flow cytometry protocol, where adherent cells are prepared for flow cytometric analysis on ice 0c, a temperature where trypsin retains activity but where intracellular kinases are inactive. In contrast, multiparameter flow cytometry of freshly drawn peripheral blood offers the promise of a highly sensitive and reproducible approach. For adherent cells lines, detach your cells from the plate, using trypsin or edta 10 mm in pbs.
The method has been tested using 6 different cell lines, 4 different assays, two different plate formats 96 and 384 well plates and two different flow cytometry instruments. This technique is most commonly used with nonadherent cells, but it can also be an effective tool for analyzing protein expression in adherent cells. Adherent cells were grown on microplates, detached with 2. Centrifuge cells 45 minutes at 2000 rpm and resuspend in the flow cytometry staining buffer so that the final cell concentration is 1x107. In a conical centrifuge tube place the cells and perform a cell count and viability analysis. Explore a clear and easy to follow stepbystep guide to flow cytometry that is wellsuited for researchers new to the technique, or experienced scientists looking for a quick refresher. After treatment with tmre, the adherent cells should be washed with pbs, trypsinized and brought to single cell suspendion. Microscopy this protocol was developed using jurkat cells treated with camptothecin to induce apoptosis and may be adapted for adherent cell lines. Centrifuge cells as in step 4 and resuspend in appropriate volume of flow cytometry staining buffer or buffer of choice so that the final cell concentration is 1 x 10 7 cells ml other cell concentrations may be appropriate for different experiments. Flow cytometric detection of most proteins in the cell. A central component of the apoptotic process is a cascade of proteolytic enzymes called caspases.
The procedures are best used for suspension cells, although some success has been achieved with adherent fibroblasts. Pbmcs isolated from whole blood through ficoll gradient centrifugation or rbc lysed whole blood or non adherent cultured cells are readily available for flow cytometric analysis. Serumstarve the cells, when needed, with serum free medium for 1216 hours prior to stimulation serum starvation lowers basal phosphorylation levels. Cells are usually stained in polystyrene round bottom 12 x 75 mm 2 falcon tubes. Phosphoprotein analysis in adherent cells using flow cytometry. Annexin v staining of adherent cells for flow cytometry for research use only. This is our basic protocol for extracellular staining of cell surface epitopes in suspension cells for flow cytometry.
Staining of cells in phosphate buffered saline pbs is not recommended. Doxorubicin, a cytotoxic agent, was administered to adherent human pancreatic carcinoma cell lines panc1 and aspc1 to produce. This is the job of the signal processing electronics. Cell surface staining of stem cell lines and other adherent cells for flow cytometry for research use only.
Wo20142142a2 method of detaching adherent cells for flow. Avoid trypsin if possible as it may damage cell surface proteins. Non adherent and loosely adherent cells in the culture supernatant can be harvested by gentle washing with pbs, and then pooled for subsequent experiments. We devised a simple twostep process for preparing adherent cells for flow cytometry with no cell loss. Collagenase or similar may be used if scraping is not sufficient for recovering adherent cells. Prepare the cells and adjust cell suspension to a concentration of 15106 cells ml. A549 cells were seeded at a density of 500,000 cellswell in 60 mm tissue culture.
Cells are more buoyant after permeabilization and much care must be excercised to maintain volume of cells. Facs is an abbreviation for fluorescenceactivated cell. The concentration also influences the rate of flow sorting, which typically progresses at 2,00020,000 cellssecond. View enhanced pdf access article on wiley online library html view. Highthroughput flow cytometry on adherent cells kaust. How do you remove the adherent cells from the flask surface. The majority of adherent cells will be macrophages, while those that. Cell surface staining of stem cell lines and other adherent. Detach adherent cells from substrate by trypsinization or. The following immunostaining protocol outlines the. This protocol bypasses washing, centrifugation, and transfer. Culture adherent cells to 80 to 90% confluence in complete medium. Cellrox green and cellrox orange flow cytometry assay kits. The concentration also influences the rate of flow sorting, which typically progresses at 2,00020,000 cells second.
As a result, ht microscopy htm is currently recommended as the method of choice when analyzing adherent cells 3. The following protocol describes a general procedure for subculturing adherent mammalian cells in culture. Several methods have been developed to quantify apoptosis, but none is adapted for all cell types. Harvest, wash the cells and adjust cell suspension to a concentration of 15 x 10 6 cells ml in ice cold pbs, 10% fcs, 1% sodium azide. This invention solves the problem of the use of adherent cell lines for medium to highthroughput screening by flow cytometry. Flow cytometry protocol flow cell signaling technology. Assessment of different permeabilization methods of. Flow cytometry protocols for surface and intracellular. Centrifuge cells as in step 4 and resuspend in appropriate volume of flow cytometry staining buffer or buffer of choice so that the final cell concentration is 1 x 10 7 cellsml other cell concentrations may. Apoptosis detection of adherent cells flow cytometry.
Caspases participate in a series of reactions that are. Protocols may to need be optimized for different cell types, targets, or applications. Fitc annexin vdead cell apoptosis kit with fitc annexin v. I understand there are many methods this could be achieved. Cell preparation for flow cytometry research use only. Twostep protocol for preparing adherent cells for highthroughput flow cytometry. Flow cytometry protocol for intracellular targets using detergents the following flow cytometry protocol for staining intracellular molecules using detergents to permeabilize cell. Flow cytometry with adherent cells purdue university. A variety of detergents and enzymes including saponin, tritonx100, tween20, np40, proteinase k, and streptolysin o were used. We have developed a simple, costeffective, and laborefficient twostep protocol for preparing adherent cells for highthroughput flow cytometry.
Trypsinize adherent cells using standard protocol nonadherent. This assay is used to count the number of cells that have undergone apoptosis. The following pdf contains useful information about the facility and sample preparation. Panels ac show example data of thp1 cells treated with controls and stained using the protocol for suspension cells. Extracellular staining for flow cytometry there are many protocols for staining cells for flow cytometry. Basically, im not sure what is the proper protocol for doing flow cytometry with adherent cells. Protocol for apoptosis assay by flow cytometry using. Cell preparation of tissue culture cells flow cytometry. Pdf twostep protocol for preparing adherent cells for. The application of ht flow cytometry htfc multiparameter analysis may, however, still be compromised due to labor intensive and time. Note that the procedure for passaging insect cells differs from that for mammalian cells on.
Remove a small amount of liquid and check it under a microscope to confirm the presence of single cells. Nih 3t3 cells were either untreated a or treated with 20m camptothecin for 24 hours b. Automated solutions for cellular analysis through flow. Trypsinize adherent cells using standard protocol non adherent cells can be counted directly. This procedure is for hela cells and some other adherent cell lines.
Flow cytometry has been extensively exploited in immunology, hematology and oncology to define cell populations via intrinsic scatter properties, cell surface antigen expression, and other fluorescence. Not for use in diagnostic or therapeutic procedures. For cells that grow in suspension, decant the cells into a conical centrifuge tube and perform a cell count and viability analysis. Apoptosis will be detected by initially staining the cells with annexin v and propidium iodide solution followed by. We describe a protocol to increase the single cell suspension yield of adherent human cells without injury. Principle of flow cytometry the basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be detected, counted and sorted. Dcfda cellular ros assay kit reactive oxygen species assay kit ab1851 uses the cell permeant reagent 2,7 dichlorofluorescin diacetate dcfda, also known as h2dcfda and as dcfhda, a. A protocol to effectively create single cell suspensions. Detection and quantification of apoptosis in transiently.
Dec 26, 2019 the application of ht flow cytometry htfc multiparameter analysis may, however, still be compromised due to labor intensive and time. Stepbystep detailed flow cytometry protocol enquire bio. Creating cell suspensions when cells are cultured under adherent conditions, they must be released from the tissue culture plate and from one anotherto. The most straight forward samples for flow cytometry are non adherent cells from tissue cell culture. I am using sirna to knockdown lamin proteins in hela cells after which i will add fitc antibodies and then follow with flow cytometry. In direct immunofluorescence staining, cells are incubated with an antibody. Aliquot 100 ul cell suspension to each 12 x 75 mm polypropylene flow cytometry tubes for a total of 106 cells per tube. Adherent cell lines, solid tissue samples, and tumors require processing into singlecell suspensions before they can be analyzed.
Preparation of cells for flow cytometry protocol for the preparation of single cells derived from tissue culture cell lines. Quantification of bacterial invasion into adherent cells by flow cytometry article in journal of microbiological methods 652. The majority of flow cytometers are now digital systems. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths. Note that the procedure for passaging insect cells differs from that for mammalian cells on several crucial steps.
Panel d shows example data for hepg2 cells treated with control compounds and stained using the protocol for adherent cells. For cells that grow in suspension, decant the cells into a conical centrifuge. E14 cells were either untreated c or treated with camptothecin for 6 hours d. Twostep protocol for preparing adherent cells for high. Finally on many occasions i have wanted to see how flow cytometry results compare with what the cells actually look like so i often culture the cells on microscope slides or on chamber slides, stain and look at them under a fluorescence or confocal microscope. Add 100 ul fixation buffer to each tube and mix by gentle vortexing. Flow cytometry protocols flow cytometry protocols ispybio. Preparation of cells for flow cytometry bioradiations.
When it comes to phospho flow cytometry on solid tissue, one. Sep 01, 2015 we have developed a simple, costeffective, and laborefficient twostep protocol for preparing adherent cells for highthroughput flow cytometry. Cells can be harvested on day 8, or step 10 can be repeated to harvest cells on day 9 or 10. For adherent cell populations, wash cells similar to a media exchange in flow cytometry staining buffer and harvest cells by gently scraping the dish, plate, or culture flask. Evaluating extracellular matrix influence on adherent cell. Spin down cell suspension at rpm for 5 minutes and decant supernatant. Annexin v staining of adherent cells for flow cytometry. Biotechniques immunostaining protocols for flow cytometric. General procedure for flow cytometry using a conjugated primary antibody. This flow cytometry ehandbook provides a brief overview of this challenging technique with numerous data examples.
This information is to serve as a guide as individual investigators may need to optimize protocols for their particular cell type cells for flow cytometry analysis are. To address this problem, we performed flow cytometry. Multicolor flow cytometry phosflow protocol for adherent cells. Flow cytometry is a popular cell biology technique that utilizes laserbased technology to count, sort, and profile cells in a heterogeneous fluid mixture. Phosflow protocol for adherent cells bd biosciencesus. Subculturing adherent cells thermo fisher scientific in. Protocols for antibody based detection page 1 of 5 psf006. Rhodamine 123 staining protocol for flow cytometry.
It is particularly difficult to reliably assay apoptosis of adherent cells. Protocol for facs analysis of hela cell transfectants. This technique is most commonly used with non adherent cells, but it can also be an effective tool for analyzing protein expression in adherent cells. Do any of you do flow cytometry on adherent cell lines. Aug 09, 2009 do you know a good method to detect apoptosis on adherent cells.
Cell surface staining of stem cell lines and other adherent cells for. Should i detach the hela cells before fixation,permeabilization,adding antibodies. Pdf twostep protocol for preparing adherent cells for high. It is important to conduct optimization experiments for every cell. Confirm the flow cytometry results by viewing the cells under a fluorescence microscope, using filters appropriate for fluorescein fitc and rhodamine tritc or texas red dye. A protocol to effectively create single cell suspensions of. Basically, im not sure what is the proper protocol for. For adherent cells lines, detach your cells from the plate.
Cell surface staining of stem cell lines and other. Examples exist where adherent cells are detached from the culturing dish by cold trypsination 12, 14, or are rather grown on microspheres 15. Phospho flow cytometry with fluorescent cell barcoding for. If so, how do you get them off the plate surface without compromising there physical properties on the cytometer.
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