Datasets for "Single-molecule and super-resolved imaging deciphers membrane behaviour of onco-immunogenic CCR5"

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