Patent Application
20180136107
The invention relates generally to the field of flow cytometry and more specifically to methods of sample collection from multi-well plates for use with flow cytometry systems.
Flow cytometry is a laser-based, biophysical technology where fluorescent molecules coupled to cells are aspirated into the flow cytometer, then passed one by one through a flow cell and excited by a set of lasers. The emitted fluorescence is then collected and transformed into an electrical signal for analysis. Labelling cells with molecules that fluoresce at different wavelengths allows the user to identify a variety of distinct cell populations and therefore it provides a powerful tool with diagnostic, therapeutic, and research applications. For example, the technique is often used to count cells from a biological sample, such as counting CD4/CD8 populations from circulating blood in human immunodeficiency virus (HIV) studies to map the disease, and to sort cells from a mixed population of cells, such as stem cell sorting from harvested biological samples for possible differentiation and reintroduction into different areas of the patient's body.
Since numerous distinct cell populations can be identified using flow cytometry often a single flow cytometry experiment or test includes a large number of samples. Therefore, there is a need to increase the throughput of flow cytometers. One solution is to present test samples on multi-well plates and program a robot to acquire samples across the multi-well plates, which is conventionally performed by aspirating cell samples from the wells. However, a challenge with presenting cells in multiwall plates is that cells tend to settle from solution and collect at the bottom of wells while waiting for aspiration, thereby decreasing cell yields in later aspirated samples.
A possible solution to the problem of cell settling is to rotate or agitate multi-well plates at various intervals to assist in suspending cells for aspiration. We attempted different intervals including between steps of aspirating samples from different wells. However, it has now been found that merely using a conventional suspension approach of rotating or agitating the multi-well plate, even at frequent intervals, only significantly helps suspend cells positioned at opposing ends of the plate. Importantly, it is found to be insufficient to suspend cells positioned within the middle region of the plates. That is, the approach tends to suspend cells in the outer region of the multi-well plate but fails to adequately suspend cells at the middle region of the plate. Accordingly, there is a need to develop an approach useful in high throughput sample acquisition for flow cytometry procedures that can increase the consistency of cell yields across the entire multiwall plate.
The above deficiencies in sample acquisition are addressed by the methods of the invention. More specifically, the methods accomplish at least two objectives related to high throughput sampling of multi-well plates in flow cytometry applications. The first is to improve consistency of particle or cell counts across the entirety of the plate. The second is to alleviate the issue of inconsistent mixing across the plate by agitation or plate shaking.
The above is achieved through a method of collecting cells from individual wells of a multi-well plate for use in a flow cytometry applications, which includes adding a suspension of cells to the wells of the multi-well plate; and aspirating cells from different wells according to a collection pattern into a flow cytometer, where the collection pattern is a sequential ordering of wells beginning at a middle region of the multi-well plate and continuing towards an outer region of the multi-well plate. In preferred embodiments the method further includes rotating or agitating the multi-well plate between steps of aspirating cells from different wells.
The method is useful for any cell or particle suitable for flow cytometry systems. Typically cells or particles will be sized between 0.2 μm to 150 μm and the methods will become still more useful as cells tend to settle from solution at higher rates. The method can be used with prokaryotic cells but is preferably used with eukaryotic cells and in particular human cells. To this end, the method can be used when detecting, counting or sorting any cell or particle population consistent with flow cytometry systems including cancer cells, monocytes, lymphocytes, and other cells isolated from tissue or grown in cultures sized between 0.2 μm to 150 μm. As known in the flow cytometry arts, the cells may be labelled with one or more labelled binding reagents against one or more cell biomarkers or exposed to one or more detectable intercalating agents. In some embodiments, the labelled binding reagents are fluorescently labelled antibodies or antibody fragments and the intercalating agent is propidium idodide. In other methods the cells are not labelled, such as experiments where only forward scatter (FSC) and side scatter (SSC) data is required.
In preferred embodiments, the multi-well plate is a 96 well plate, characterized as having rows A-H and columns 1-12. Preferably, the middle region is characterized as a well selected from the group consisting of wells D6, D7, E6 and E7. In some embodiments, cells from wells B4-B8, C4-C8, D4-D8, and E4-E8 are aspirated before cells from remaining all wells in a same 96 well plate. Still further, in some embodiments, cells from wells C5-C7, D5-D7, and E5-E7 are aspirated before cells from all remaining wells of a same 96 well plate. In embodiments where two or more plates are provided, the methods may aspirate samples from the above wells from different plates prior to aspirating cells from all remaining wells of the different plates or may aspirate all wells from a same plate prior to aspirating cells from a different plate.
In some embodiments, the collection pattern is a spiral-square configuration, which is characterized as a series of wells in successive square patterns that each circle the middle region and where the sequential ordering proceeds by aspirating adjacent wells. When the multi-well plate is a 96 well plate, the spiral-square configuration can include three successive square patterns around a central well, such as well selected from the group consisting of wells D6, D7, E6 and E7 but preferably D6. In some embodiments, the spiral-square configuration is further characterized as successively aspirating cells from wells that are immediately adjacent to the prior well that was aspirated for at least one half of all wells in the 96 well plate.
In an exemplary embodiment, the multi-well plate is a 96 well plate defined by rows A-H and columns 1-12, and the sequential ordering includes the well order D6, E6, E7, D7, C7, C6, C5, D5, E5, F5, F6, F7, F8, E8, D8, C8, B8, B7, B6, B5, B4, C4, D4, E4, F4, G4, G5, G6, G7, G8, G9, F9, E9, D9, C9, B9, A9, A8, A7, A6, A5, A4, A3, B3, C3, D3, E3, F3, G3, H3, H4, H5, H6, H7, H8, H9, H10, G10, F10, E10, D10, C10, B10, A10. In one variation, the order further includes the order of A2, A1, B1, B2, C2, C1, D1, D2, E2, E1, F1, F2, G2, G1, H1, H2, H11, H12, G12, G11, F11, F12, E12, E11, D11, D12, C12, C11, B11, B12, A12, A11. In another variation, the order further includes meandering or alternating between rows of column 1 and column 2 or rows of column 11 and column 12.
In other embodiments the collection pattern is a nearest well to center pattern, which is characterized as successively aspirating cells from a well that is nearest to the middle region, where the middle region is defined as a center of the multi-well plate independent of whether a well is positioned at the center. A modified nearest well to center is a variation on the embodiment and is characterized as successively aspirating cells from a well that is nearest to both the middle region and a well from which an immediately prior cell was aspirated.
In another exemplary embodiment the multi-well plate is a 96 well plate and the collection order is D6, E6, E7, D7, C7, C6, D5, E5, F6, F7, E8, D8, C8, C5, F5, F8, E9, D9, B7, B6, D4, E4, G6, G7, G8, F9, C9, B8, B5, C4, F4, G5, H6, H7, E10, D10, A7, A6, D3, E3, G4, G9, B9, B4, C3, F3, H5, H8, F10, C10, A8, A5, A4, B3, G3, H4, H9, G10, B10, A9, D2, E2, E11, D11, C11, F11, F2, C2, A3, H3, H10, A10, B11, G11, G2, B2, D1, E1, E12, D12, C12, F12, F1, C1, A2, H2, H11, A11, B12, G12, G1, B1, A1, H1, H12, and A12.
The collection pattern may include two or more distinct patterns. In some embodiments the first pattern is a square pattern or spiral-square pattern and a second pattern is a meandering pattern.
The collection patterns themselves may be programmed by the user into a sample collection or acquisition software module or can be preloaded in flow cytometer as one or more presets, which provides the user with a selectable option to use one or more sample collection patterns. Alternatively, a sample collection module of sample acquisition software may automatically perform the collection patterns. The software may also assign the rotation or shaking interval of the multi-well plate.
In a related embodiment, the invention provides a method of collecting cells from individual wells of a multi-well plate for flow cytometry, the method comprising determining particle or cell counts from each well of the multi-well plate by flow cytometry; ordering the wells from lowest particle count to highest particle count; generating a sequential order for collection that follows the ordering of wells to establish a collection pattern; and collecting particles or cells according to the collection pattern in a second multi-well plate by flow cytometry. In preferred embodiments, the method also includes rotating or agitating the multi-well plate between steps of collecting particles or cells from different wells. In some embodiments the pattern exactly follows the ordering of wells from lowest particle count to highest particle count; however, in other embodiments the collection pattern deviates from the ordering of wells less than 15%.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the invention but instead to demonstrate the improvements over the state of the art and various exemplary embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows.
The term “multi-well plate” as used herein refers to a plate having a plurality of wells capable of retaining a population of cells having a suitable working volume for flow cytometry sample aspiration. Multi-well plates are commonly available in formats of 6 well, 12 well, 24 well, 48 well, 96 well, 384 well and 1536 well and can be flat bottom or rounded bottom. Conventional 6 and 12 well plates have working volumes of about 1.9-2.9 mL and 0.76-1.14 mL respectively and are not typically used for highthroughput flow cytometry. Conventional 48 well plates have a working volume of about 190-285 μL and therefore may be used but are not typically preferred due to the desire to increase the number of available wells thereby increasing throughput. Conventional 96 well plates have a working volume of about 100-200 μL and are preferred as they balance the needs of high throughput with sufficient sample volume for high throughput flow cytometry. Conventional 384 well plates having a working volume of 25-50 μL and may be suitable but much less preferred as the sample volumes are on the low end of flow cytometry applications. 1536 well plates having a working volume of 5-10 μL and are therefore much less desirable. Most preferably, the multi-well plate is a 96 well plate having a rounded bottom.
The term “row-by-row” as used herein refers to a collection pattern that proceeds across a single row, then proceeds across a neighboring row, and so on until all rows are aspirated. In the case of a conventional 96 well plate, cells or particles from wells assigned to column 1-12 of the row “A” are typically aspirated first, followed by the aspiration of cells or particles from wells assigned to columns 1-12 of row “B”, then row “C”, and so on until aspirating samples from all columns across each row.
The term “column-by-column” as used herein refers to a collection pattern that proceeds up or down a single column, then proceeds up or down the neighboring column and so on. In the case of a 96 well plate, samples from each row of the column conventionally referred to as column “1” are typically aspirated first, followed by the aspiration of cells or particles from each row of column “2”, then column “3”, and so on until cells are aspirated from all rows of each column.
The term “spiral-square” as used herein is a collection pattern that follows a sequence of successive square patterns in a spiral order. The spiral order can be a right handed spiral or a left handed spiral, which spirals to the right or left.
The term “sequential ordering” as used herein refers to the order of wells for aspiration.
The term “middle region” as used herein refers to the center most point of the multi-well plate together with immediately adjacent wells. Preferably, the middle region is characterized as having four (4) or fewer wells. If there are an even number of rows or columns, such as on a 96 well plate, the middle region can be the combination of wells that form the center of both the rows and columns (e.g. a region defined by D6, D7, E6, and E7 on a 96 well plate), any two of the combination of wells (e.g. a region defined by one of D6 and D7, E6 and E7, D6 and E6, D6 and E7, E6 and E7) or any single well of the combination (e.g. D7, D7, E6, or E7).
The term “continuing towards” as used herein refers to a progression over a series of wells.
The term “successive pattern” as used herein refers a pattern of wells that maintains a prior pattern of wells but differs in size. “Successive square pattern” in a 96 well plate can include a first square pattern formed from wells E5-E7, C7-E7, C5-C7 and C5-E5; followed by a second square pattern formed from wells F4-F8, B8-F8, B4-B8, and B4-F4; and followed by a third square pattern formed from wells G3-G9, A9-G9, A3-A9 and A3-G3.
The term “immediately adjacent” as used herein refers to the positioning of two wells next to one another without an intervening well.
The term “well that is nearest to the center of the multi-well plate and from which an immediately prior cell was aspirated” as used herein to the well that has not yet been aspirated and that compared to remaining wells that have not yet been aspirated has the lowest sum of distances to the both the center of the multi-well plate and to the well from which a sample was last aspirated.
The term “selectable preset” as used herein refers to a software option that encodes the collection pattern that may be selected by a user without additional programming.
The term “biomarker” as used herein refers to a detectable moiety of a cell. A biomarker may be a cluster of differentiation (CD) for a cell, such as a glycoprotein found on a surface of a cell, can be a nucleic acid, or any other moiety useful for detecting one or more cells in flow cytometry.
As an introduction to the invention, among the challenges presented during sample acquisition in high throughput flow cytometry systems that use multi-well plates is maintaining consistent particle or cell counts across all wells of the plate. In particular, as cells wait for aspiration they tend to settle to the bottom of the well. While one of ordinary skill in the art may consider rotating or agitating the plate using a rotating platform or shaking platform, Example I demonstrates this does not adequately suspend cells across the entirety of the multi-well plate. More specifically, Example I and corresponding
In Example I, cells were added in equal amounts to each well across the multi-well plate. Wells were aspirated, and the plate was rotated or agitated between aspirating each well. The collection pattern used was a conventional a row-by-row sample collection method, where samples were collected across row A before proceeding to row B and so on. There was a significant discrepancy found in particle counts across the wells. That is, particle counts were found to be inconsistent across the plate when using a row-by-row approach even with rotating or agitating the plate after aspirating each well. In particular, as shown in
As shown in
Although the examples demonstrate the improvements over the art with respect to particles and in particular to silica beads or polystyrene beads, the method is equally useful for cell analysis. The similarity of tested 5 μm polystyrene beads (Spheroteach Accucount Beads) to cells is depicted in
The skilled artisan will also appreciate that cells can be harvest from any suitable fluid or organ as known in the flow cytometry arts, such as blood, serum or tissue biopsy. Cells can be harvested from adipose tissue. Alternatively, the cells can be from cell lines as known in the art to which the invention belongs. To this end, the method can be used when detecting, counting or sorting any cell or particle population consistent with flow cytometry systems including cancer cells, monocytes, lymphocytes, and any other cells isolated from tissue or grown in cultures sized between 0.2 μm to 150 μm and suited for flow cytometry applications.
Although the methods herein significantly improve counts across the entire multi-well plate and significantly improve counts from the middle region of the multi-well plate, the methods may be optimized to achieve even greater consistency in particle or cell accounts. Among these modifications the user may wish to optimize the number of cells added to each well. That is, the artisan may find it beneficial to add fewer or more cells across the multi-well plate, although it is generally preferred to have the same number of cells in each well. Another modification may be to alter the volume in the well such as increasing the sample volume or decreasing the sample volume, which may depend on the concentration of cells in the well.
In some instances, cells will be added to the wells, aspirated according to the collection pattern and analyzed according to forward scatter (FSC) or side scatter (SSC) parameters. Such comparisons can be used to isolate monocyte populations from red blood cells. However, the skilled artisan will also appreciate that since flow cytometry often involves labelling cells with one or more binding reagents for fluorescent detection, the method will be especially useful in such procedures. Accordingly, prior to aspiration the cells may be labelled with one or more labelled binding reagents against one or more cell biomarkers or exposed to one or more detectable intercalating agents. Consistent with the flow cytometry arts, the labelled binding reagents can be fluorescently labelled antibodies or fluorescently labelled antibody fragments. There are many fluorescent labels from which the artisan may choose, of which flourescein (FITC), phycoerythrin (PE) and allophycocyanin (APC) tend to be very popular. However, research and development groups are continually developing new fluorochromes for the flow cytometry arts and thus the availability of corresponding labelled binding reagents are increasingly available through a variety of companies, including but not limited to Becton Dickenson (Franklin Lakes, N.J.), Jackson ImmunoResearch Laboratories, Inc (West Grove, Pa.) and many others known those skilled in the flow cytometry arts. Each of these can be used with the present invention. In some embodiments the intercalating agent is propidium idodide, which can intercalate into DNA molecules. Again, such molecules are also commonly known in the art to which the invention belongs and are included in the present invention.
In flow cytometry, cells are aspirated using an aspirating needle, which directs cells into the flow cytometer, where cells mix with sheath fluid and are streamed through a flow cell for excitation. As such, the invention is directed towards maximizing the availability of cells through the flow cell by provide new sample collection patterns that increase the consistency of cell counts or particle accounts across the multi-well plate. The term “collection pattern” as used herein refers to the sequence or order for aspirating wells across the multi-well plate for collection by the flow cytometer and thus streaming through the flow cell.
In some embodiments, the collection pattern may be ordered beginning with wells having a tendency to produce a lowest particle or cell count to wells having a tendency to produce a highest particle or cell count. This order can be determined experimentally by counting cells or particles across the multi-well plate over a series of experiments while agitating or rotating the plate. The wells can then be statistically ordered from lowest particle or cell count to highest particle or cell count and the order designated as a collection pattern for future sample collection.
Alternatively, the collection pattern can include groups of wells that are grouped according to count ranges then ordered such that groups with lower count ranges are aspirated before groups with higher count ranges. For example, a group of wells having a count range of 0-250 counts would precede a group of wells with a count range of 250-500 counts, which would precede a group of wells with a count range of 500-1000 counts. This approach may be preferred when count variation between wells within a same count range continually alter the aspiration sequence using an absolute lowest to highest ordering. That is, wells in a same region, such as the middle most region, may equally suffer from low cell or particle counts and therefore their relative ordering with each other may be less important compared to their ordering compared to wells of different regions on the multi-well plate.
In some embodiments, the collection pattern is a series of successive square patterns, where a first square pattern of wells is encircled by a second square pattern of wells, and so on. In such a pattern the order of aspirating wells in each square pattern may be randomly assigned or may be further ordered such as ordered clockwise or counterclockwise.
In some embodiments, the collection pattern is a spiral-square pattern, which is characterized as a series of successive square patterns that circle the middle region or a central well with a caveat that moving to a next well in the collection sequence is performed by aspirating a well that is adjacent (either horizontally adjacent, vertically adjacent, or diagonally adjacent) to the most recently aspirated well. An example is shown in
The artisan will also appreciate that the above collection pattern can also be applied to multi-well plates having more or less than 96 wells. In such cases, the middle-most well can be chosen or chosen from a group of middle most wells, and the collection pattern followed.
In some instances a square collection pattern or spiral-square collection pattern cannot be followed throughout the entire multi-well plate because of the plate layout. That is, in some instances, such as that of
In an exemplary embodiment, the multi-well plate is a 96 well plate and the collection order follows the wells D6, E6, E7, D7, C7, C6, C5, D5, E5, F5, F6, F7, F8, E8, D8, C8, B8, B7, B6, B5, B4, C4, D4, E4, F4, G4, G5, G6, G7, G8, G9, F9, E9, D9, C9, B9, A9, A8, A7, A6, A5, A4, A3, B3, C3, D3, E3, F3, G3, H3, H4, H5, H6, H7, H8, H9, H10, G10, F10, E10, D10, C10, B10, A10. In one variation, the order further comprises A2, A1, B1, B2, C2, C1, D1, D2, E2, E1, F1, F2, G2, G1, H1, H2, H11, H12, G12, G11, F11, F12, E12, E11, D11, D12, C12, C11, B11, B12, A12, A11. In another variation, the order further comprises a second collection sequence alternating or meandering between column 1 and column 2 or column 11 and column 12.
Another embodiment is depicted in
An exemplary embodiment of a nearest to center collection pattern is shown in
As with prior embodiments, the nearest to center collection pattern can be combined with a second pattern that differs from the first. In such embodiments, preferably the nearest to center collection pattern is followed over the first 25% of wells, more preferably over first 50% of wells and still more preferably of the first 75% of wells.
Similarly, the symmetry of a multi-well plate may permit the collection pattern to proceed to the left or to the right, each of which is encompassed herein.
In other embodiments a collection pattern deviates less than 15% from one of the collection patterns disclosed, where the deviation from a collection pattern is determined by comparing the collection sequences and calculating the percent difference. If the percent difference is within 15 percent it is within the corresponding collection pattern.
Since collection patterns themselves are sets of ordered sequences, the collection patterns may be programmed by the user into a sample collection software module for use with a flow cytometer or can be provide as one or more collection presets in the flow cytometer, which provides the user with a selectable option to use one or more collection patterns. Alternatively, the sample collection module of sample acquisition software may automatically perform the collection patterns herein.
Collection patterns have been followed by adapting a currently available multi-sampling flow cytometry system, namely, the NOVOCYTE flow cytometer offered by ACEA BIOSCIENCES, INC. together with a sample collection software program.
Depending on the adjustability of the user's flow cytometer system, the artisan may follow the disclosed patterns of sample collection to initiate suitable collection pattern programming. In other embodiments, software designers may preprogram software to collect samples according the patterns disclosed herein and optionally provide the user with one or more selectable options or patterns for sample collection. The skilled artisan will appreciate that the programming of collection patterns herein is not limited to any particular program language as programming could differ between flow cytometry systems. In some embodiments, the programming may adapt a Cartesian coordinate system where plots along perpendicularly aligned axes correspond to well locations.
Embodiments of the invention may be further understood in light of the following examples, which are not to be construed as limiting the scope of the disclosure in any way.
The inventions addresses inconsistencies in cell counts found to continually occur when using a row by row or column by column based method in sample acquisition. The following example demonstrates the inconsistencies found in wells across a 96 well plate when measuring cell counts using a row by row sample acquisition approach using the NOVOCYTE flow cytometer (ACEA Biosciences, Inc., San Diego, Calif.).
A suspension of silica beads was vortexed to ensure the beads were equally distributed throughout the solution. An equal volume of the suspension was aliquoted into each well of a 96 well plate to ensure an equal load across all wells. The NOVOCYTE flow cytometer (ACEA BIOSCIENCES, San Diego, Calif.) was programmed to sequentially acquire 10 uL of sample from each well and count the silica beads in each acquired sample. Between acquiring samples from each well, the plate was subjected to rotation or agitation for 15 seconds using a shaking platform. The pattern of sample acquisition followed a row by row sequence, which acquires samples sequentially from columns 1-12 of row A, followed by acquiring samples sequentially from columns 1-12 of row B, followed by acquiring samples sequentially from columns 1-12 of row C, followed by acquiring samples sequentially from columns 1-12 of row D, followed by acquiring samples sequentially from columns 1-12 of row E, followed by acquiring samples sequentially from columns 1-12 of row F, followed by acquiring samples sequentially from columns 1-12 of G, and finally followed by acquiring samples sequentially from columns 1-12 of row H.
The total counts for each well were recorded and are reproduced in
As shown in
The following example demonstrates improved consistency in particle counts across the multi-well plate when acquiring samples using a spiral-square approach.
A suspension of silica beads was vortexed to ensure the beads were equally distributed throughout the solution. An equal volume of the suspension was aliquoted into each well of a 96 well plate to ensure an equal load across all wells. The NOVOCYTE flow cytometer (ACEA BIOSCIENCES, San Diego, Calif.) was programmed to sequentially acquire 10 uL of sample from each well and count the silica beads in each acquired sample. Between acquiring samples from each well, the plate was subjected to rotation or agitation for 15 seconds. The pattern of sample acquisition followed a spiral-square configuration using the following order. D6, E6, E7, D7, C7, C6, C5, D5, E5, F5, F6, F7, F8, E8, D8, C8, B8, B7, B6, B5, B4, C4, D4, E4, F4, G4, G5, G6, G7, G8, G9, F9, E9, D9, C9, B9, A9, A8, A7, A6, A5, A4, A3, B3, C3, D3, E3, F3, G3, H3, H4, H5, H6, H7, H8, H9, H10, G10, F10, E10, D10, C10, B10, A10, A2, A1, B1, B2, C2, C1, D1, D2, E2, E1, F1, F2, G2, G1, H1, H2, H11, H12, G12, G11, F11, F12, E12, E11, D11, D12, C12, C11, B11, B12, A12, A11.
The total counts for each well were recorded and are reproduced in
As shown in
The following example demonstrates improved consistency in particle counts across the multi-well plate when acquiring samples using a nearest well to center approach.
A suspension of silica beads was vortexed to ensure the beads were equally distributed throughout the solution. An equal volume of the suspension was aliquoted into each well of a 96 well plate to ensure an equal load across all wells. The NOVOCYTE flow cytometer (ACEA BIOSCIENCES, San Diego, Calif.) was programmed to sequentially acquire 10 uL of sample from each well and count the silica beads in each acquired sample. Between acquiring samples from each well, the plate was subjected to rotation or agitation for 15 seconds. The pattern of sample acquisition followed a nearest well to center configuration using the following order. D6, E6, E7, D7, C7, C6, D5, E5, F6, F7, E8, D8, C8, C5, F5, F8, E9, D9, B7, B6, D4, E4, G6, G7, G8, F9, C9, B8, B5, C4, F4, G5, H6, H7, E10, D10, A7, A6, D3, E3, G4, G9, B9, B4, C3, F3, H5, H8, F10, C10, A8, A5, A4, B3, G3, H4, H9, G10, B10, A9, D2, E2, E11, D11, C11, F11, F2, C2, A3, H3, H10, A10, B11, G11, G2, B2, D1, E1, E12, D12, C12, F12, F1, C1, A2, H2, H11, A11, B12, G12, G1, B1, A1, H1, H12, A12.
The total counts for each well were recorded and are reproduced in
As shown in
Since the above examples demonstrated improved consistency across the entire plate compared to the row-by-row approach, the spiral-square approach was also tested with reduced loads.
A suspension 5 μm polystyrene beads (Spherotech Accucount Beads) was vortexed to ensure the beads were equally distributed throughout the solution. An equal volume of the suspension was aliquoted into each well of a 96 well plate to ensure an equal load across all wells. The NOVOCYTE flow cytometer (ACEA BIOSCIENCES, San Diego, Calif.) was programmed to sequentially acquire 10 uL of sample from each well and count the silica beads in each acquired sample. Between acquiring samples from each well, the plate was subjected to rotation or agitation for 15 seconds. The pattern of sample acquisition followed a spiral-square configuration using the following order. D6, E6, E7, D7, C7, C6, C5, D5, E5, F5, F6, F7, F8, E8, D8, C8, B8, B7, B6, B5, B4, C4, D4, E4, F4, G4, G5, G6, G7, G8, G9, F9, E9, D9, C9, B9, A9, A8, A7, A6, A5, A4, A3, B3, C3, D3, E3, F3, G3, H3, H4, H5, H6, H7, H8, H9, H10, G10, F10, E10, D10, C10, B10, A10, A2, A1, B1, B2, C2, C1, D1, D2, E2, E1, F1, F2, G2, G1, H1, H2, H11, H12, G12, G11, F11, F12, E12, E11, D11, D12, C12, C11, B11, B12, A12, A11.
The total counts for each well were recorded and are reproduced in
As shown in
The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of exemplary improvements of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the invention. Such modifications are also intended to fall within the scope of the appended claims.
This application is a divisional of U.S. patent application Ser. No. 14/750,613 filed Jun. 25, 2015, which claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. provisional patent application Ser. No. 62/017,251, filed Jun. 25, 2014; the content of each is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62017251 | Jun 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14750613 | Jun 2015 | US |
| Child | 15870661 | US |
