A Hemocyte Disturbance Assay to Assess Hemocyte Re-Adhesion to Hematopoietic Pockets

Abstract

Source: Petraki, S. et al., Assaying Blood Cell Populations of the Drosophila melanogaster Larva. J. Vis. Exp. (2015)

This video demonstrates the hemocyte disturbance assay, evaluating hemocyte re-adhesion in Drosophila larvae's hematopoietic pockets. Mechanical disturbance releases resident hemocytes, elevating circulating hemocyte counts. Post-recovery, re-adherence of resident hemocytes is confirmed, illustrating reversibility.

Protocol

1. Hemocyte Bleed/Scrape Assay Preparation of slides: Option 1 for microscopes without tile scanning function: For each larva to be analyzed, prepare one glass slide with about 5 Pap-pen wells of 2 mm squares each, corresponding to the field viewing area of the microscope; add approximately 5 – 10 µl of S2 media to each (Figure 1A). Keep slides in a moist chamber to prevent wells from drying out. Option 2 for microscopes with tile scanning function…

Representative Results

Figure 1. Hemocyte Bleed/Scrape and Disturbance Assay setup and schematic. (A) Single Image Slide Setup: five 2mm squares for imaging with a 5X objective. (B) Tile Scan Slide Setup: four 3 mm squares for imaging bleed/scrapes of ≤2.5 mm larvae with a tile scan microscope. Recommended objectives for imaging are 5X or 10X. (C) Bleed/S…

Materials

6cm/9cm Petri dishes			One for each genotype to be evaluated
Water squirt bottle
Metal spoon/spatula
Thin paintbrush			e.g. a "liner"
Glass cavity dish
PAP pen: Super PAP PEN IM3580	Beckman Coulter
Glass slides			Each slide will have 5 or more PAP PEN squares drawn on them. Size of squares depends on the imaging objective and magnification of the microscope camera; e.g. 2mm squares.
Moist chamber			This will be used to prevent slides and wells from drying out: sealed container with wet paper towels lining the sides/bottom
Schneider's Drosophila cell culture media	Invitrogen
Cold block			This is a metal block (a.k.a. heating block) chilled in bucket containing ice; preferably black-colored or other dark, non-reflective color
Two 1ml syringes with needles (27G ½)	Becton Dickinson		For dissections.
Optional: Surgical spring scissors (cutting edge 2mm)	Fine Science Tools
Glass beads, 212-600 micron	Sigma
2 ml Eppendorf tubes	Eppendorf		One per genotype evaluating
Vortex Mixer	Fisher Scientific
Transgenic Drosophila larvae with fluorescently marked hemocytes. Suitable transgenes include: HmlΔ-DsRed (Makhijani et al., 2011), MSNF9mo-mCherry (Tokusumi et al., 2009), BcF6-CFP and -GFP (Gajewski et al., 2007), or HmlΔ-GAL4 (Sinenko and Mathey-Prevot, 2004), Pxn-GAL4 (Stramer et al., 2005), He-GAL4 (Zettervall et al., 2004), Crq-GAL4 (by H. Agaisse (Stramer et al., 2005)), or eater-GAL4 (Tokusumi et al., 2009) combined with UAS-GFP or other fluorescent protein transgenes.
Fluorescence dissecting microscope	Leica		Here: Leica M205, optional with camera, imaging software and measuring module
Inverted fluorescence microscope with camera attachment	Leica or Keyence		With or without tile scanning function (eg. Leica DMI series, Keyence BIOREVO BZ-9000 series)