Datasets for "Single-molecule and super-resolved imaging deciphers membrane behaviour of onco-immunogenic CCR5"
Authors/Creators
- 1. University of York
- 2. National Physical Laboratory, UK
Description
Flow cytometry
Modality / instrument: Flow cytometer (CytoFLEX LX, Beckman Coulter)
File format: FCS + XIT (CytExpert, Beckman Coulter).
Samples and acquisitions:
Fluorescent fusions in live Chinese Hamster ovary (CHO) cells.
|
File |
Cell line |
Runs |
Cells counted |
|
CONTROL.fcs |
CHO wild-type |
1 |
7000 |
|
GFP-CCR5.fcs |
CHO-GFP-CCR5 |
1 |
7000 |
|
Exp_20220916_1_GFP.xit |
N/A - metadata |
Approx. size 6 MB
PaTCH microscopy images
Imaging modality / instrument: Brightfield + PaTCH fluorescence microscopy
Image format: OME TIFF (16 bit) + MicroManager metadata files
Microscope settings:
488 nm triggered excitation; split red/green detection, cropped to green (GFP) channel only; 10 ms/frame laser exposure; 13.5 ms/frame-to-frame; 53 nm/px. Photometrics Prime95b CMOS.
Samples and acquisitions:
Fluorescent fusions of GFP-CCR5 receptor in live CHO cells imaged with and without 100 nM CCL5 ligand. Each subfolder corresponds to a field of view and contains one brightfield and one PaTCH acquisition of the same cell.
|
Folder |
Condition |
Fields of view |
|
AC6 CONTROL sc |
CCL5- |
11 |
|
AC6 CCL5 sc |
CCL5+ (100 nM) |
10 |
Approx. size before compression: 14 GB
Structured illumination microscopy - volumetric stacks
Imaging modality / instrument: SIM fluorescence microscopy (custom setup at NPL based on Olympus IX71)
Image format: OME TIFF (16 bit) with intrinsic metadata (voxel size)
Microscope settings: 638 nm excitation; 60x/1.3 NA; Flash 4.0, Hamamatsu Photonics. For additional details see the reference below (Hunter et al, bioRxiv).
Samples and acquisitions:
Dylight 650-MC-5 labeled CCR5 receptor in fixed CHO-CCR5 cells, imaged with and without 100 nM CCL5 ligand. Each acquisition is of a unique field of view and contains one SIM reconstruction as an XYZ volumetric stack. ‘Basal membrane’ acquisitions consist of 5 slices at 200 nm z-intervals across the range of the basal membrane. ‘Whole cell' acquisitions are made up of 7 slices with 500 nm z-interval ranging from just below the basal membrane to just above the apical membrane.
| Folder | Subfolder/condition | Fields of view |
| Basal membrane | CCL5- | 5 |
| Basal membrane | CCL5+ (100 nM) | 6 |
| Whole cells | CCL5- | 5 |
| Whole cells | CCL5+ (100 nM) | 8 |
Approx. size before compression: 300 MB
Files
Flow cytometry (CHO-GFP-CCR5 and CHO wt).zip
Additional details
Funding
- UK Research and Innovation
- Mechanistic Biology and its strategic application BB/T007222/1
- UK Research and Innovation
- Developing next generation bioimaging/biophotonics tools to dissect the immunological synapse in single cells, one molecule at a time 2279374
- UK Research and Innovation
- Biological physics of protein clustering in epigenetic memory and transcriptional control EP/T002166/1
References
- Single-molecule and super-resolved imaging deciphers membrane behaviour of onco-immunogenic CCR5. Patrick Hunter, Alex L. Payne-Dwyer, Michael Shaw, Nathalie Signoret and Mark C. Leake. bioRxiv 2022.05.19.492692v1; doi: https://doi.org/10.1101/2022.05.19.492692