The Photoconversion Technique for Exploring Inflammatory Cell Dynamics in Insect Pupae
Published: January 31, 2024
Abstract
Source: Weavers, H. et al., Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae. J. Vis. Exp. (2018)
The video demonstrates the use of photoconversion to study inflammatory cell dynamics in Drosophila pupae. Green hemocytes are drawn to the wounded epithelium, followed by distant migration. Photoconversion shifts certain hemocytes from green to red, facilitating their movement tracking.
Protocol
This protocol consists of four main sequential steps: (1) Preparation of Drosophila stocks and staging of Drosophila pupae, (2) Pupal dissection and mounting, (3) Pupal wounding, (4) in vivo time-lapse confocal imaging. 1. Preparation of Drosophila Stocks and Staging of Pupae Obtain appropriate Drosophila stocks (see Introduction and Table of Materials). …
Representative Results
Figure 1: Drosophila pupa preparation for wounding and live-imaging. (A) Drosophila white prepupae collected at 0 h APF, with the anterior end indicated by everted breathing appendages (spiracles). (B) After raising white 0h APF prepupae for 18 h at 25 °C, the puparium appears brown. Dissection of the pupal case should b…
Declarações
The authors have nothing to disclose.
Materials
| Drosophila stocks | |||
| Ubiquitous GFP-tagged E-cadherin;Ubi-p63E-shg.GFP; (chrII) | Kyoto Stock Center, DGRC | #109007 | Ubi-p63E promoter sequences drive the expression of Drosophila E-cadherin (shotgun) tagged at the C-terminal end with GFP. |
| Ubiquitous GFP-tagged E-cadherin;;Ubi-p63E-shg.GFP (III) | Bloomington Drosophila Stock Centre (Indiana University) | #58742 | Ubi-p63E promoter sequences drive the expression of Drosophila E-cadherin (shotgun) tagged at the C-terminal end with GFP. |
| Ubiquitous GFP-tagged Moesin P{sGMCA}3.1 | Bloomington Drosophila Stock Centre (Indiana University) | #59023 | The ubiquitously expressed sqh promoter/enhancer drives expression of a fragment of Moesin (that includes the actin binding sequences) tagged with GFPS65T. |
| Hemocyte specific serpent-Gal4 driver ;srp-Gal4; | Generated by Katja Bruckner | Generated by Katja Bruckner | The expression of ScerGAL4 fused to a polyA tail is controlled by 2 genomic sequences from upstream of Drosophila serpent. Ref: Brückner, K., Kockel, L., Duchek, P., Luque, C.M., Rørth, P., Perrimon, N. The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev Cell. 7 (1), 73–84, doi: 10.1016/j.devcel.2004.06.007 (2004). |
| UAS-nuclearRFP w1118;;P{UAS-RedStinger}6 | Bloomington Drosophila Stock Centre (Indiana University) | #8545 or #8547 | UAS regulatory sequences drive expression of the DsRed.T4 form of RFP which is tagged at the C-terminal end with a nuclear localisation signal |
| UAS-cytoplasmicGFP ;;P{UAS-GFP.S65T} | Bloomington Drosophila Stock Centre (Indiana University) | Multiple stocks available (e.g. #1522) | Expression of the S65T version of GFP by UAS regulatory sequences; the S65T variant exhibits increased brightness. |
| UAS-photoconvertibleKaede w1118;; P{UAS-Kaede.A}3 | Bloomington Drosophila Stock Centre (Indiana University) | #26161 | Kaede protein emits bright green fluorescence after synthesis, but changes efficiently to a bright stable red fluorescence on irradiation with UV. |
| GFP-tagged spaghetti squash w1118;;P{sqh-GFP.RLC} | Bloomington Drosophila Stock Centre (Indiana University) | #57145 | The sqh coding region, which is tagged at the C-terminal end with a T:AvicGFPS65T tag, is expressed under the control of the natural sqh promoter. |
| Ingredients for fly food media | Fly food media is made according to standard procedures (see Greenspan, R. 1997. Fly Pushing: The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Press. 1-191 pp.) | ||
| Maize | Wild Oats, Bristol, UK (or equivalent supplier) | Contact supplier direct | organic |
| Soya flour | Wild Oats, Bristol, UK (or equivalent supplier) | Contact supplier direct | organic |
| Malt extract | Wild Oats, Bristol, UK (or equivalent supplier) | Contact supplier direct | organic |
| Molasses | Wild Oats, Bristol, UK (or equivalent supplier) | Contact supplier direct | organic |
| Difco agar | BD Biosciences, Fisher Scientific | DF0142-15-2 | For preparation of fly food |
| Propionic acid | Sigma | 402907 | For preparation of fly food |
| Nipagen | Sigma | 79721 | For preparation of fly food |
| Dried baker's yeast | Redstar, Dutscher Scientific, UK LTD | Redstar, Dutscher Scientific, UK LTD | For preparation of fly food |
| Sample preparation and mounting | |||
| Parafilm | Sigma | P7793-1EA | For preparation of heptane glue |
| Fine sable paintbrush | Daler-Rowney (or equivalent) | #0 or 1 | |
| Forceps | Fisher Scientific (or Fine Science Tools) | NC9404145 | Dumont #5 |
| Glass bottomed dishes for imaging | MatTek | P35G-0-10-C | We suggest using 35mm petri dishes, with at least a 10mm Microwell, 0.085-0.13mm cover glass, uncoated. Dishes with larger microwells will enable increasing numbers of pupae to be mounted and imaged in a single experiment. |
| Heptane | Sigma | 51730-5ML | For preparation of heptane glue |
| Double sided sticky tape (e.g. Scotch) | Agar Scientific | AGG263 | For preparation of heptane glue |
| 50ml tube (for heptane glue) | Falcon tubes from Fisher Scientific | 14-432-22 | For preparation of heptane glue |
| Glass microscope slides | Agar Scientific | AGL4244 | For dissection of Drosophila pupae |
| Dissecting stereo microscope with brightfield | Leica (or equivalent) | M50 | For dissection of Drosophila pupae |
| Microscissors | John Weiss International | 103123 | Miniature Research Scissors (straight) |
| Laser ablation and imaging | |||
| Nitogen ablation laser | Spectra-Physics (or Andor equivalent) | Model VSL-337ND-S | For wounding, this should be attached to a widefield imaging system |
| Multilaser confocal laser-scanning microscope (CLSM) | Leica (or equivalent) | TCS AOBS SP8 or SP5-II attached to a Leica DMi8 inverted epifluorescence microscope (or equivalent) | Ideally including a motorised stage for multi-site and 'mosaic' scanning, plus 'hybrid' GaAsP detectors (that offer much greater sensitivity and boosting of low signal) |
| Environmental chamber | Life Imaging Services (or equivalent) | "Microscope Temperature Control System" | Attached to Confocal microscope for temperature control during imaging |
| Image Analysis Software | |||
| FRAP software module | Leica (or equivalent) | CLSM FRAP software module | For performing photoconversion of photoconvertible fluorophores such as Kaede |
| ImageJ (image analysis software) | National Institutes of Health (NIH) | https://imagej.nih.gov/ij/ | Schneider, C.A., Rasband, W.S., Eliceiri, K.W. "NIH Image to ImageJ: 25 years of image analysis". Nature Methods 9, 671-675, 2012. |
| ImageJ plugin "Manual Tracking" | National Institutes of Health (NIH) | https://imagej.net/Manual_Tracking | |
| ImageJ plugin "TrackMate" | ImageJ, NIH | https://imagej.net/TrackMate | Tinevez, JY.; Perry, N. & Schindelin, J. et al. (2016), "TrackMate: An open and extensible platform for single-particle tracking.", Methods 115: 80-90, PMID 27713081 |
| Volocity (high performance 3D imaging software) | Perkin Elmer | Volocity 6.3 | For image analysis |
| IMARIS (image analysis software) | Bitplane | IMARIS for Cell Biologists | For image analysis |
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The Photoconversion Technique for Exploring Inflammatory Cell Dynamics in Insect Pupae. J. Vis. Exp. (Pending Publication), e21878, doi: (2024).