A549 cells -- High-throughput Secretomic Analysis of Single Cells - Dataset (ID:20121)
|HMS Dataset ID:||20121|
|Dataset Title:||A549 cells -- High-throughput Secretomic Analysis of Single Cells|
|Publication(s) Using Dataset:||PMID: 23339603|
|Screening Lab Investigator:||Rong Fan|
|Screening Principal Investigator:||Rong Fan|
|Assay 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.|
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.|
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.
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.
4. The antibody glass slide is put on the top of PDMS microwell array with antibody barcode resting on the cell capture chambers.
5. Then the PDMS microwell array and glass slide are clamped tightly with screws and pressure is distributed by springs.
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.
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.
8. After imaging, the assembly is put into an incubator at 37 °C for 24 hrs to allow for cell secretion.
9. After the trapped cells are incubated for 24 hours, the screws are released to remove the antibody barcode glass slide.
10. ELISA immunoassay procedures are performed. ELISA procedures are followed to translate secreted cytokines by single cells into detectable signals.
11. The results are detected and analyzed with Genepix scanner (e.g., Genepix 4200A) and software (e.g., Genepix 7.0).
12. The fluorescence results are then matched to each of the chambers of the sub-nanoliter microchamber array chips analyzed via optical imaging.
Cell count: number of cells captured per micro-chamber. Cell count of 1 indicates that 1 cell is captured.
Data columns: fluorescence intensity for each antibody-labeled protein. Note that negative values occur due to background subtraction.
Each record (row) represents the secretion profile for a single micro-chamber.
|HMS Dataset Type:||Microfluidics|
|Date Publicly Available:||2013-06-10|
|Most Recent Update:||2016-08-24|