Anyaporn Sawasdichai and Nazefah Abdul Hamid are grateful to the Royal Thai Government and the Government of Malaysia respectively for Ph.D. Scholarships. This work was funded by a Wellcome Trust project grant awarded to PSJ and KG. We are also grateful to the University of Bristol Wolfson Bioimaging Facility.

I. Preparation for fractionation
This section describes the preparation of poly-L-Lysine coated microscope coverslips and the attachment of cells prior to fractionation. If required the cells can be transiently transfected with protein expression vectors either before or after attachment.
A. Preparation of poly-L-Lysine coated coverslips
<ol>
	<li>Prepare a solution of 1mg/ml poly-L-Lysine in distilled water.</li>
	<li>Coat clean coverslips with poly-L-Lysine by incubating them in the solution for at least 1 hour on a rocking platform at 22&deg;C.</li>
	<li>Wash the coated coverslips with sterile distilled water twice and follow with a single wash with 96% ethanol.</li>
	<li>Air dry the coated coverslips on a piece of filter paper and keep them in a dry container for future use (once dry they can be stacked).</li>
</ol>
B. Attaching cells to coverslips
Non-adherent cells (here we use K562 cells)
<ol>
	<li>Place a poly-L-Lysine coated coverslip in a well in a 6-well plate with the coated surface facing up.</li>
	<li>Seed K562 cells at a density of 7x105 cells/well in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and PS (penicillin 100units/ml, streptomycin 100mg/ml). In the case of K562 cells transient transfection can be performed using electroporation (0.4cm electroporation cuvettes with 1x107 cells in 200&mu;l of media at 250V / 975&mu;F) before seeding the cells.</li>
	<li>Incubate the cells for 24 hours at 37&deg;C in 5% CO2.</li>
	<li>Pour off the medium and wash the cells twice with ice cold phosphate buffered saline (PBS).</li>
	<li>Follow with the subcellular fractionation protocol.</li>
</ol>
Adherent cells (here we use Cos-7 cells)
<ol>
 <li>Place an uncoated coverslip in a well in a 6-well plate.</li>
 <li>Wash twice in PBS prewarmed at 37&deg;C.</li>
 <li>Detach the cells by digesting with trypsin (0.03% EDTA, 0.25% trypsin) in PBS at 37&deg;C for 2 to 5 minutes. It is important not to over-digest the cells and to stop the digestion as soon as predominantly individual floating cells are present.</li>
 <li>Stop the digestion by seeding the cells at a density of 3x105 cells/well in a 6-well plate in DMEM supplemented with 10% FBS and PSG (penicillin 100 units/ml, streptomycin 100ug/ml and L-glutamine 292ug/ml), on normal coverslips. Adherent cells generally attach well onto normal microscope coverslips, however, poly-L-Lysine coated coverslips can be used if required.</li>
 <li>Incubate the cells for 24 hours at 37&deg;C and 5% CO2. In the case of Cos-7 cells transient transfection can be performed using Fugene 6 (Roche) if required and the cells left to recover for a further 24 hours at 37&deg;C and 5% CO2.</li>
 <li>Pour off the medium and wash the cells twice with ice cold PBS.</li>
 <li>Follow with the subcellular fractionation protocol.</li>
</ol>
 
II. Subcellular fractionation
In situ fraction is performed according to the schematic shown in Figure 1 and is based on a method described previously5 with modifications described previously7 and in the detailed protocol below. It is recommended to transfer the coverslips to a fresh 6-well plate following each fractionation step removing the coverslip before removing the liquid. Although only four coverslips are required for imaging, additional coverslips are required to produce whole cell extract and nuclear matrix fractions for western blotting if this is to be performed in parallel. 
<ol>
	<li>Prepare and filter cytoskeleton buffer (CSK): 10mM PIPES, 300mM Sucrose, 100mM NaCl, 3mM MgCl2, 1mM EGTA. CSK buffer should be freshly prepared on the day of fractionation.</li>
	<li>If required the cells from one coverslip can be used to prepare whole cell extract for western blotting by gently placing 200ul TES buffer (1% SDS, 2mM EDTA, 20mM Tris-HCl pH 7.4) directly onto the coverslip and incubating for 1 or 2 minutes at 22&deg;C. The whole cell extract can then be removed and the proteins precipitated by adding 250ul of 1M (NH4)2SO4 and incubating on ice until the other western samples are ready.</li>
	<li>Wash the remaining coverslips with 1ml ice cold PBS twice at 22&deg;C by tilting the 6 well-plate two or three times.</li>
	<li>Remove the coverslip representing whole cells and transfer to a fresh 6-well plate. Follow with the cell fixation and immuno-fluorescence protocol.</li>
	<li>Gently remove the PBS from the remaining wells.</li>
	<li>Extract the cytoplasmic and loosely held nuclear proteins by gently placing 200ul CSK buffer + 0.1% (V/V) Triton X-100 directly onto each remaining coverslip and incubating on ice for 1 minute.</li>
	<li>Remove the cytoplasmic and loosely held nuclear extract and precipitate as described in step 2.</li>
	<li>Wash the coverslips with 1ml ice cold PBS twice at 22&deg;C by tilting the 6 well-plate.</li>
	<li>Remove the coverslip representing tightly held nuclear material and follow with the cell fixation and immuno-fluorescence protocol.</li>
	<li>Gently remove the PBS from the remaining wells then extract the tightly held proteins by gently placing 200ul CSK buffer + 0.5% (V/V) Triton X-100 directly onto each remaining coverslip and incubating on ice for 20 minutes.</li>
	<li>Remove the tightly held extract and precipitate as described in step 2.</li>
	<li>Wash the coverslips with 1ml ice cold PBS twice at 22&deg;C by tilting the 6 well-plate.</li>
	<li>Remove the coverslip representing chromatin fraction and follow with the cell fixation and immuno-fluorescence protocol.</li>
	<li>Gently remove the PBS from the remaining wells then place 200ul CSK buffer + 100ug/ml of DNase I onto the remaining coverslips and incubate for 30 minutes at 37&deg;C.</li>
	<li>Remove the chromatin extract and precipitate as described in step 2.</li>
	<li>Wash the coverslips with ice cold PBS twice at 22&deg;C by tilting the 6 well-plate.</li>
	<li>Remove the coverslip representing the nuclear matrix fraction and follow with the cell fixation and immuno-fluorescence protocol.</li>
	<li>If required the matrix proteins can be removed from an additional matrix coverslip for western blotting using 200ul TES buffer as described in step 2.</li>
	<li>Samples for western blot should be centrifuged at 13000 rpm in a bench top microcentrifuge at 4&deg;C and the pellets resuspended in 40ul SDS loading buffer (62.5mM Tris-HCl pH 6.8, 2% SDS (w/v), 50 mM DTT, 10% glycerol, 0.01% bromophenol blue(w/v)) before analysis by SDS-PAGE. <img src="/files/ftp_upload/1958/1958fig1.jpg" alt="Figure 1" /> Figure 1. A schematic representation of in situ subcellular fractionation. Cytoskeleton buffer (CSK) consists of 10mM PIPES, 300mM Sucrose, 100mM NaCl, 3mM MgCl2, 1mM EGTA. TES buffer consists of 1% SDS, 2mM EDTA, 20mM Tris-HCl pH 7.4.</li>
</ol>
III. Cell fixation and immuno-fluorescence
<ol>
 <li>Gently add 1ml of 4% formaldehyde/PBS to each coverslip and incubate at 4&deg;C for 30 minutes.</li>
 <li>Wash each coverslip gently 2-3 times with 1ml of PBS at 22&deg;C.</li>
 <li>Add 400ul of 0.1% Triton X-100/PBS to each coverslip and incubate at 22&deg;C for 10 minutes.</li>
 <li>Wash each coverslip gently 2-3 times with 1ml of PBS at 22&deg;C.</li>
 <li>Add 400ul of 3% Bovine Serum Albumin (BSA)/PBS and incubate at 22&deg;C for 20 minutes to reduce potential background staining.</li>
 <li>Wash each coverslip gently 2-3 times with 1ml of PBS at 22&deg;C.</li>
 <li>Place 100ul of the primary antibody in PBS directly onto the coverslip and incubate at 22&deg;C for 1 hour. Primary and secondary antibodies should be diluted in PBS to the required concentration. This may need to be optimized for each antibody used.</li>
 <li>Wash each coverslip gently 2-3 times with 1ml of PBS at 22&deg;C.</li>
 <li>Place 200ul of secondary antibody in PBS and incubate at 22&deg;C away from light for 1 hour.</li>
 <li>Wash each coverslip gently 2-3 times with 1ml of PBS at 22&deg;C.</li>
 <li>Mount each coverslip (cells down) onto a glass microscope slide using Vectashield mounting medium containing DAPI (4',6-diamidino-2-phenylindole hydrochloride).</li>
 <li>Wipe excess mounting medium from around the coverslip using a paper towel and incubate the slide at 22&deg;C for at least 30 minutes away from light.</li>
 <li>Fix the coverslip to the glass slide using clear acrylic nail polish.</li>
 <li>Keep the slides away from light before visualization by fluorescence microscopy.</li>
</ol>
IV. Representative results
<img src="/files/ftp_upload/1958/1958fig2.jpg" alt="Figure 2" /> Figure 2. Subcellular fractionation of non-transfected COS-7 cells. Fractionated Cos-7 cells were fixed with formaldehyde and immunostained using &alpha;-Tubulin, &alpha;-Histone H1 or &alpha;-Lamin A/C antibodies conjugated to TRITC followed by treatment with mounting medium containing DAPI. Images were acquired with 10x ocular lens and 63x objective lens using a Leica confocal microscope. Cell nuclei were visualized using a DAPI filter set while in the same field cells stained with marker antibodies were visualized using a TRITC filter set. 
<img src="/files/ftp_upload/1958/1958fig3.jpg" alt="Figure 3" /> Figure 3. A GFP-6E2 fusion protein is associated with the nuclear matrix. Subcellular fractionation of Cos-7 cells expressing a fusion protein consisting of green fluorescent protein (GFP) fused to the human papillomavirus (HPV) type 6 E2 protein (GFP-6E2). Cell nuclei were visualized via DAPI staining while localization of GFP-6E2 in the transfected cells was directly visualized via GFP fluorescence. Lamin A/C was visualized using &alpha;-Lamin A/C antibodies conjugated to TRITC and a TRITC filter set. The merge images reveal that some of the GFP-6E2 protein is associated with the nuclear matrix (lower right panel). Images were obtained as described in Figure 2.

<ol>
	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

No conflicts of interest declared.

Common problems and suggestions:
All or most of the cells are lost during washing. During washing steps liquids must be added slowly to the side of the 6-well plate avoiding the coverslip. Similarly liquids should be removed by carefully tilting the plate and slowly pipetting off excess fluid. Cell adhesion can be increased using poly-L-Lysine coated coverslips.
Genomic DNA is not completely digested. For some cell types the DNAse I digestion step may need to be exte...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
<td>&nbsp;</td>
<tr>
<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EGTA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>E4378-25G </td>
<td>&nbsp;</td>
<tr>
<td>Fomaldehyde</td>
<td>Chemical</td>
<td>BDH</td>
<td>284216N</td>
<td>&nbsp;</td>
<tr>
<td>PIPES</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Chemical</td>
<td>Fisher Scientific</td>
<td>S/8600/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Chemical</td>
<td>Sigma</td>
<td>T-6876</td>
<td>&nbsp;</td>
<tr>
<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Histone H1</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-8030</td>
<td>&nbsp;</td>
<tr>
<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
<td>&nbsp;</td>
<tr>
<td>Tubulin-&alpha;</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-32293</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in situ subcellular fractionation of adherent and non-adherent mammalian cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

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15.6K Views.  University of Bristol. In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Video: In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...