Improved method for processing autoradiographs of cells grown on multiwell plates

A modification of an established procedure for autoradiographic processing of cultured cells is described. This method eliminates the need for pipetting each individual well and also for cutting and dismantling the multiwell plate for slide preparation. In this procedure the entire plate can be processed as a single unit and the cells can be analyzed in situ, thus eliminating the time consuming pipetting and cutting procedures. Furthermore, the entire experiment can be filed without use of additional slides or storage boxes. Hence, this is a simpler, time conserving, and economical way to process large numbers of cultures for thymidine labeling indices.     

Freezing human ES cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines. A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction. Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature. During this time cells can be observed rounding, and colonies lifting off the plate surface. Gentle repeated pipetting will remove cells and colonies from the plate surface. Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun. Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10 cm plate. After cells are adequately resuspended, ice cold freezing media is added at equal volume. Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80 C over 24 hours. Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.     

Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA

We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.     

The core-microtome: A new tool for surface preparation on cores and time series analysis of varying cell parameters

A microtome designed for the surface preparation of entire increment cores allows cutting plane surfaces on cores up to a length of 40 cm. Compared to the common sanding procedure, the wood cells of the annual rings remain open, not filled with swarf, and the cell walls are smooth and hence clearly visible. This article aims at describing the functionality of the microtome and the procedures needed for an accurate surface preparation to achieve a good contrast for subsequent image analysis. Possible applications for a more detailed analysis of variations in the tracheid structure of conifers and vessel sizes of oak are presented, which can be included in time series analyses.     

High throughput gene expression measurement with real time PCR in a microfluidic dynamic array

We describe a high throughput gene expression platform based on microfluidic dynamic arrays. This system allows 2,304 simultaneous real time PCR gene expression measurements in a single chip, while requiring less pipetting than is required to set up a 96 well plate. We show that one can measure the expression of 45 different genes in 18 tissues with replicates in a single chip. The data have excellent concordance with conventional real time PCR and the microfluidic dynamic arrays show better reproducibility than commercial DNA microarrays.     

Coated microwell plate-based affinity purification of antigens

Antibody-based affinity capture of antigens is widely used in the isolation of antigens from complex mixtures. Antibody and the corresponding antigen are allowed to interact with each other to form immunocomplexes which are then typically captured by protein A or protein G immobilized on beaded support. Antigen capture performed using this method generally requires multiple centrifugation steps and careful pipetting to avoid loss of the bead-bound complexes. This traditional procedure is tedious and not easily reproducible, especially when working with multiple samples. To address these issues we have demonstrated that antigens can be captured with protein A/G, protein G, and high binding-capacity streptavidin 96-well strip-coated plates. The coated plate method of antigen purification is reproducible, within the same experiment and between experiments, due to the uniform binding capacity of the plates and wells. Here we report the use of coated microwell plates for antigen capture and for protein-protein interaction studies with the well-characterized BIR2-SMAC, transferrin receptor/ transferrin, and other systems.     

Rapid isolation of single malaria parasite-infected red blood cells by cell sorting

Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum-infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.     

Rapid cloning of mammalian cells with honeycomb cloning plates and nonlethal vital stains

A rapid and technically simple method for cloning both adhesive and nonadhesive mammalian cells is described. The procedure employs (a) honeycomb cloning plates and (b) nonlethal vital stains. Instead of placing cloning rings around colonies, cells are initially seeded at clonal density directly into a plate containing an array of cloning rings (the honeycomb plate). Hence, the time involved in placing cloning rings around colonies is eliminated. Second, clone-containing wells of the honeycomb plate are easily identified by staining plates with the nonlethal vital stains, MTT or INT tetrazolium. Vital staining eliminates the time involved in searching for clones. Last, clones are transferred with a cotton-tipped swab thereby eliminating the time involved in trypsinization of cells. In this fashion, one can pick and transfer clones of substrate adherent mammalian cells at a rate of one clone/10 to 15 s. Thus, mammalian cells can be cloned as rapidly as cloning can be carried out in microbial systems.     

Semiautomated preparation of DNA templates for large-scale sequencing projects

The rate limiting step in a large-scale sequencing project is the generation of single-stranded DNA templates. We describe a fast, semiautomated procedure, using 96-well microtitre plates, in which 192 templates can be readily prepared in 1 day. The technique can be carried out manually or can be semiautomated using a robot pipetting device. We also provide evidence for the reliability and applicability of this method to a large-scale sequencing project.     

Rapid cloning of mammalian cells with honeycomb cloning plates and nonlethal vital stains

Summary A rapid and technically simple method for cloning both adhesive and nonadhesive mammalian cells is described. The procedure employs (a) honeycomb cloning plates and (b) nonlethal vital stains. Instead of placing cloning rings around colonies, cells are initially seeded at clonal density directly into a plate containing an array of cloning rings (the honeycomb plate). Hence, the time involved in placing cloning rings around colonies is eliminated. Second, clone-containing wells of the honeycomb plate are easily identified by staining plates with the nonlethal vital stains, MTT or INT tetrazolium. Vital staining eliminates the time involved in searching for clones. Last, clones are transferred with a cotton-tipped swab thereby eliminating the time involved in trypsinization of cells. In this fashion, one can pick and transfer clones ofsubstrate adherent mammalian cells at a rate of one clone/ 10 to 15 s. Thus, mammalian cells can be cloned as rapidly as cloning can be carried out in microbial systems. This study was supported, in part, by Grant CA 33074 from the National Cancer Institute, Bethesda, MD, Grant PCM-8218137 from the National Science Foundation, Washington, D.C., and a grant from the National March of Dimes.     

Effect of cutting the zona pellucida on the pronuclear transplantation in the mouse

A new and reliable pronuclear transplantation procedure for the mouse egg has been developed by McGrath and Solter ('83). To overcome the technical difficulties of such a procedure, especially in uniformly preparing enucleation pipettes and in reducing damages during micromanipulation, we have examined the effect of cutting the zona pellucida of the eggs. By making a slit in the zona of an egg, the time for pipetting and exchange of pronuclei between eggs was shortened because the sharp tip of the pipette was not necessary. Although the proportion of pregnant recipients and young obtained after transfer of pronuclear transplanted eggs cultured for 1 day or 3 days was quite low, it was significantly increased (70% for pregnancy rate and 32% for the young) following transfer of eggs cultured for 4 days. These values were comparable with those after transfer of unoperated eggs cultured to morulae and blastocysts.     

Simultaneous Histological Preparation of Bone, Soft Tissue and Implanted Biomaterials for Light Microscopic Observations

Block specimens of formalin fixed bone, soft tissue and endosseous implanted biomaterials can be successfully embedded in polymethyl methacrylate by employing vacuum desiccation during the dehydration steps and refrigeration during the infiltration step. One-hundred-micrometer histological sections can be obtained from the cured polymethyl methacrylate blocks by cutting with a low concentration diamond wafering blade on a Buehler Isomet Circular Low Speed Saw using Buehler Isocut fluid. The sections can be readily stained and details of individual cells studied by light microscopy, thus allowing interpretation of the relationship between biomaterial and surrounding tissues. The advantage of this method is that it allows observation of the entire specimen in situ. The details of the procedure are presented.     

The application of flow cytophotometry in measurements of cell adhesion

A common approach to the study of cell substrate interactions is the measurement of the attachment of cells to different substrates or to cultured cell layers. The evaluation of attachment is made either by scintillation counting of previously labelled adhering cells, or by light microscopy using the criterion of cell shape, sometimes refined by automatic image analysis. These methods have many drawbacks. This paper suggests the use of fluorescence-activated flow cytophotometry, (FC) which yields direct counts of the non-adhering cells. These "free" cells are removed after completion of the adhesion experiment from the microtitre plate wells. An internal standard, in the form of fluorescent polystyrene beads is added, allowing evaluation of the percentage of cells adhering to the well walls. Flow cytophotometry then produces data based on the analysis of large populations of cells. Unequivocal discrimination is obtained between the counted cells and counted fluorescent beads eliminating counting errors. The results can be processed on line by computer. A suspension of mouse splenocytes was used for the evaluation of the overall error of the method arising from inaccuracies in pipetting, interference of glutaraldehyde with ethidium bromide (EB) staining and instrumental error. Each adhesion experiment was terminated by staining and post-fixation and it was established that this introduces no change in cell counting, in comparison with the original unfixed cells. Prefixation, however, quenches the EB staining and would interfere with the counting procedure. The overall standard error of the technique was found to be 5%-10%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.     

Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template

A simple, rapid method is presented for the enzymatic sequence analysis of nanogram amounts of single-stranded or double-stranded DNA. This approach employs the thermostable DNA polymerase from Bacillus sterothermophilus and exploits its ability to efficiently extend all of the template-primer complex, even at low substrate concentrations. The procedure requires few pipetting steps, no preannealing step and very short reaction time. This method can significantly reduce the cost associated with DNA polymerase and the amount of template and time required to perform the enzymatic sequencing reactions. As little as a 10-ng aliquot of such sequencing reactions can be analyzed on a fluorescence-based capillary gel electrophoresis instrument recently developed in our laboratory. This highly sensitive detection, in conjunction with the ability to efficiently sequence nanogram amounts of template, strongly suggests the feasibility of direct DNA sequencing of single bacteriophage M13 plaques without prior amplification.     

Rapid sample processing and fast gas chromatography for identification of bacteria by fatty acid analysis

A commercially available system for microbial identification by fatty acid analysis (Microbial Identification System (MIS), MIDI, Newark, DE, USA) requires a four-step sample derivatization procedure in screw-cap test tubes. By using glass tubes in a 96-well format with multichannel pipetting, the time required for sample preparation can be greatly reduced. The standard gas chromatography column, 25 m long by 0.20 mm ID, is replaced with a 10 m long by 0.10 mm ID column, reducing the gas chromatography run time to one third of the standard time. Either or both of these procedures can be easily implemented in any laboratory using the MIDI system, resulting in faster identifications and higher sample throughput.     

Delay of intracellular calcium transients using a calcium chelator: application to high-throughput screening of the capsaicin receptor ion channel and G-protein-coupled receptors.

Whole-cell functional assays are often used for high-throughput screening (HTS) of molecular targets such as ion channels and G-protein-coupled receptors. A common method for assaying the activity of these membrane proteins is to measure the change in intracellular calcium concentration upon receptor stimulation. These changes in calcium concentration are typically transient and therefore not readily adapted to high-density plate formats used in HTS instruments. We have demonstrated that an intracellular calcium chelator, BAPTA, was able to delay by 5- to 20-fold and extend for several minutes the observed calcium signals initiated by extracellular calcium influx or release of calcium from intracellular stores. As examples, we used cells expressing a calcium-permeable ion channel, vanilloid receptor type 1 (the capsaicin receptor), and two G-protein-coupled receptors. These receptor-mediated increases in intracellular calcium concentration were measured by both fluorescence-based and luminescence-based detection methods. The use of an intracellular calcium chelator to delay calcium signaling should have wide application since it allows the measurement of the functional activity of any cellular receptor that signals through calcium. With this procedure, calcium fluorescence and luminescence whole-cell functional assays may be performed with standard laboratory pipetting and detection systems.     

A method for removing the brain and spinal cord as one unit from adult mice and rats

To collect complete rodent spinal cord samples for histological analysis, researchers typically use a method that involves fixation of the carcass, followed by decapitation and removal of the vertebrae and the spinal cord. Researchers then decalcify, process and embed the spinal column in paraffin. When this method is used, the spinal cord retains its natural curvature, which may be undesirable to some investigators. The authors describe a methodology by which the entire spinal cord, with the brain attached, can be removed from a mouse or rat, set against a rigid support material and fixed perfectly straight. This allows for more precise sectioning and simplified histological analysis. Researchers can even create block preparations, each of which contains multiple spinal cord sections, so that they can compare anatomically matched sections. This procedure can also be used to obtain fresh spinal cord samples that are free of bone and can be frozen in optimal cutting temperature medium.     

Enabling high‐throughput biology with flexible open‐source automation

Our understanding of complex living systems is limited by our capacity to perform experiments in high throughput. While robotic systems have automated many traditional hand‐pipetting protocols, software limitations have precluded more advanced maneuvers required to manipulate, maintain, and monitor hundreds of experiments in parallel. Here, we present Pyhamilton, an open‐source Python platform that can execute complex pipetting patterns required for custom high‐throughput experiments such as the simulation of metapopulation dynamics. With an integrated plate reader, we maintain nearly 500 remotely monitored bacterial cultures in log‐phase growth for days without user intervention by taking regular density measurements to adjust the robotic method in real‐time. Using these capabilities, we systematically optimize bioreactor protein production by monitoring the fluorescent protein expression and growth rates of a hundred different continuous culture conditions in triplicate to comprehensively sample the carbon, nitrogen, and phosphorus fitness landscape. Our results demonstrate that flexible software can empower existing hardware to enable new types and scales of experiments, empowering areas from biomanufacturing to fundamental biology.     

High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence

An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-gamma by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-gamma secreted from NK3.3 cells and employed it in high throughput screening for IFN-gamma production inhibitors. With this screening format, IFN-gamma can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.