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Dataset Information
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Dataset Name:
U87 cells -- High-throughput Secretomic Analysis of Single Cells

Dataset Description:
This micro-fluidics assay is performed using a microchip developed by the Yale LINCS U01 team. This microchip consists of a PDMS microwell array containing >5000 single-cell trapping chambers and a high-density antibody barcode array fabricated by Yale LINCS flow patterning method. The assay does not require external fluid handling equipment and can be performed anywhere in a typical cell biology lab.

--Data in Package:
20122.txt

--Metadata in Package:
Cell_Line_Metadata.txt

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Center-specific Information
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Center-specific Name:
HMS_LINCS

Center-specific Dataset ID:
20122

Center-specific Dataset Link:
http://lincs.hms.harvard.edu/db/datasets/20122/

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Assay Information
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Assay Protocol:
1. Before performing the single cell trapping experiment, the PDMS microwell array and antibody barcode glass slide is blocked with 3% BSA solution (Sigma) respectively for 2 hrs and then rinsed with fresh cell medium.<br />
2. Cells are suspended in fresh medium just before cell capture. The PDMS microwell array is placed facing upward and cell culture medium solution is removed until a thin layer is remained on the PDMS microwell array surface.<br />
3. Cell suspension is pipetted (50-200 uL) onto the microwell array and allowed to settle for 10 mins so that cells would fall into the microwells.<br />  
4. The antibody glass slide is put on the top of PDMS microwell array with antibody barcode resting on the cell capture chambers.<br />
5. Then the PDMS microwell array and glass slide are clamped tightly with screws and pressure is distributed by springs.<br /> 
6. Single cells will be trapped in the microwell array and the assembly is allowed to incubate for 24 hours to allow for cell secretion.<br /> 
7. The assembly is imaged on an automatic microscope stage to acquire optical images (Nikon Ti phase contrast imagine with a motorized stage) recording the number and location of cells in each microwell.<br />
8. After imaging, the assembly is put into an incubator at 37 C for 24 hrs to allow for cell secretion.<br /> 
9. After the trapped cells are incubated for 24 hours, the screws are released to remove the antibody barcode glass slide.<br />
10. ELISA immunoassay procedures are performed. ELISA procedures are followed to translate secreted cytokines by single cells into detectable signals.<br /> 
11. The results are detected and analyzed with Genepix scanner (e.g., Genepix 4200A) and software (e.g., Genepix 7.0).<br />
12. The fluorescence results are then matched to each of the chambers of the sub-nanoliter microchamber array chips analyzed via optical imaging.<br />
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Cell count: number of cells captured per micro-chamber. Cell count of 1 indicates that 1 cell is captured.<br />
Data columns: fluorescence intensity for each antibody-labeled protein. Note that negative values occur due to background subtraction.<br />
Each record (row) represents the secretion profile for a single micro-chamber. <br />

Date Updated:
2016-08-24

Date Retrieved from Center:
11/13/2015

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Metadata Information
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Metadata information regarding the entities used in the experiments is included in the accompanied metadata. A metadata file per entity category is included in the package. For example, the metadata for all the cell lines that were used in the dataset are included in the Cell_Lines_Metadata.txt file.
Descriptions for each metadata field can be found here: http://www.lincsproject.org/data/data-standards/
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