Materials and Reagents

Falcon tubes (15 ml) (Corning, Falcon^®, catalog number: 352196 )
Eppendorf tubes
Cover glass, No 1.5, 22 mm² (Corning, catalog number: 2850-22 )
Petri dish (100 x 15 mm) (Corning, catalog number: 351029 )
Kimwipe
Micro slides (Corning, catalog number: 2948-75X25 )
Sterile filter unit: 0.22 µm cellulose acetate filter flasks (Corning, catalog number: 430769 )
Drosophila melanogaster wandering third instar larvae (Ashburner, 1989)
E. coli (DH5α) constitutively expressing GFP
1. peGFP (https://www.addgene.org/vector-database/2485/)
2. pET-GFP-C11 (http://www.addgene.org/30183/)
Bucket of ice
LB growth media (Fisher Scientific, catalog number: BP1425-500 )
Schneider’s Drosophila medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 21720 ) with 10% FBS (Atlanta Biologicals, Advantage, catalog number: S11095 )
S2 cell media
PBSS = 0.3% Saponin (Sigma-Aldrich, catalog number: S7900 ) in PBS
10% NGS
Phalloidin Alexa 594 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A12381 )
Vectashield (Vector Laboratories, catalog number: H-1000 )
Clear nail polish
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O) (Sigma-Aldrich, catalog number: S9390 )
Potassium phosphate monobasic (KH₂PO₄) (Fisher Scientific, catalog number: P285 )
Hydrochloric acid (HCl)
Sodium hydroxide (NaOH)
Paraformaldehyde (Electron Microscopy Sciences, catalog number: 19208 )
10% normal goat serum (Jackson ImmunoResearch, catalog number: 005-000-121 ) in PBSS
10x PBS (see Recipes)
8% paraformaldehyde (see Recipes)
Fixative: 4% paraformaldehyde in PBS (see Recipes)

Equipment

Spectrophotometer (Molecular Devices, model: SpectraMax M2 )
Low speed centrifuge for 15 ml Falcon tubes (Eppendorf, model: 5804 R )
Dissecting stereomicroscope with Leica L2 cold light source (Leica Microsystems, model: Leica L2 )
Fine dissecting forceps (Fine Scientific Tools)
37 °C incubator with shaking (Eppendorf, New Brunswick^TM, model: Innova^® 44 )
25 °C incubator (BioCold Environment, model: BC49-IN )
Dissecting dish
Confocal Microscope
Note: We use a Zeiss LSM510 (Zeiss, model: LSM510 ) with 63 x NA1.4 objective.

Software

ImageJ (NIH)
Prism (GraphPad)

Procedure

Preparation of GFP-expressing E. coli
1. Inoculate 50 ml LB broth with a GFP E. coli colony.
2. Incubate overnight at 37 °C with shaking.
3. Use spectrophotometer to measure optical density at 600 nm (OD₆₀₀).
  1. Measure bacteria with broth, and broth alone.
  2. Subtract OD₆₀₀ of fresh LB broth from bacteria with broth.
4. Dilute bacteria to OD₆₀₀ = 0.1
5. Re-suspend bacteria in Schneider’s Drosophila medium with FBS
Primary hemocyte isolation
1. Warm fresh Schneider’s Drosophila medium to 25 °C.
2. Collect ten wandering third instar larvae in a large drop of water on dissecting dish–wash thoroughly and place in fresh drop.
3. Place a 22 mm² coverslip in a 100 x 15 mm Petri dish.
4. Add 200 μl Schneider’s Drosophila medium from step B1 to the coverslip.
  1. This should create a large droplet.
  2. Keep the area of the droplet as small as possible.
  3. Do not let the droplet spread out across the coverslip.
5. Place the washed larvae into the large droplet on the coverslip.
6. Under a dissecting microscope, use forceps to pinch and immobilize the tail end of the larva and use another forceps to grab and rip the outer cuticle of the larvae from tail to mouth (see Figure 1).
  
  Figure 1. Larval dissection. This image depicts the way forceps are used to hold (left forceps) and dissect the cuticle of larvae covered with S2 cell media with the right forceps along the larval midline (jagged black line) thereby releasing the larval hemolymph into the S2 cell media.
7. Rip open all ten larvae in the media droplet on the coverslip.
  Note: Do this step as quickly as possible.
8. Remove larval carcasses from the media droplet.
9. Incubate hemolymph in media on coverslip in the Petri dish to allow hemocytes to attach to coverslip (at 25 °C for 15 min).
  1. This 15-min incubation will be sufficient time for hemocytes to adhere to the coverslip.
  2. After about 10 min, melanization may begin to occur (see Figure 2).
  3. Begin washes with PBS if the media features small black spots. These indicate the beginning of melanization that we seek to avoid.
  4. Do not allow the media to turn black.
    
    Figure 2. Early signs of melanization. The image displays a coverslip (22 x 22 mm) with a drop of medium containing hemocytes settling on the coverslip. Over time, crystal cells in the medium will initiate melanization that can be detected by the dark spots in the solution (arrow) and the dark ring at the edge of the drop (arrowhead). At this stage, hemocytes should be washed with PBS to remove crystal cells.
10. Pour 1x PBS carefully into the Petri dish away from the coverslip to avoid dislodging the adherent cells.
  1. Wash thoroughly by slowly swirling the dish.
  2. Pour off PBS from Petri dish.
  3. Repeat wash (B10).
11. Hemocytes will adhere to the coverslip, while the PBS washes remove the majority of crystal cells, which are mainly responsible for melanization.
12. Remove the coverslip with forceps and place on Kimwipe, then place in a dry Petri dish on ice.
Expose hemocytes to GFP bacteria
1. Add enough GFP bacteria in Schneider’s Drosophila medium from step A5 (40,000 bacteria/µl) to cover the entire coverslip without spilling over the edge of the coverslip (~200 µl).
2. Incubate bacteria with hemocytes on ice for 20 min to allow the bacteria to adhere to the cell surface.
3. Rinse 3 x by pouring PBS into the Petri dish and swirling gently. Remove PBS by pouring off.
4. Remove coverslip with forceps and place on Kimwipe, then place in dry Petri dish.
5. Add fresh, warm Schneider’s Drosophila medium on the coverslip for the desired amount of chase time (in wild-type hemocytes the majority of bacteria will be digested and undetectable by 45 min).
Fix, stain, and mount hemocytes (all performed at room temperature)
1. Wash the media with PBS as before.
2. Add enough 4% paraformaldehyde to cover the coverslip without spilling over its edges.
3. Fix cells for 30 min.
4. Thoroughly wash cells with PBS as before.
5. Add PBSS for 30 min to permeabilize cell membranes.
6. Remove PBSS and add 10% NGS in PBSS for 30 min to block unspecific antibody binding.
7. Stain with primary and secondary antibodies of your choice and/or Phalloidin-Alexa 594 in PBSS for one hour.
  Note: Keep specimen in the dark as much as possible during and after secondary antibody staining.
8. Wash with PBS 3 x 15 min after each antibody.
9. Place the coverslip on Kimwipe to dry it.
10. Add a small drop of Vectashield to a microscope slide.
11. Slowly lay the coverslip with stained hemocytes onto the drop of Vectashield.
12. Seal the edges of the coverslip to the slide with nail polish.
13. Image the cells on a confocal microscope with a 63x objective and 3x digital zoom.

Data analysis

To determine the number of remaining bacteria at different time points in different genetic backgrounds, confocal images were opened with ImageJ (NIH) and smoothed with a Gaussian blur of one before merging bacteria and phalloidin-stained channels. The number of bacteria within individual cells was counted and recorded in a Prism (GraphPad) spreadsheet as a single data point for each cell. Bacteria were counted when the entire GFP bacterium was surrounded by phalloidin staining. At least 25 cells were counted for each of three experiments. Prism software was used to plot a box and whisker graph and to perform a one-way ANOVA comparing relevant data sets (all to all).

Notes

The experiment described above has been beneficial to our lab’s analysis of bacterial accumulation and phagosome maturation within primary hemocytes (Figure 3) and the effect of different genotypes (Akbar et al., 2011; Rahman et al., 2012; Akbar et al., 2016). Chase time and antibody conditions can be adapted for a wide variety of experimental questions. However, great care must be taken during the initial steps of hemocyte isolation from larvae. As mentioned above, the Schneider’s Drosophila medium cell media droplet must maintain a dome-like shape in order to contain the larvae within the droplet and to keep hemocytes in a defined area with ample nutrients.

Figure 3. Micrograph of hemocyte and GFP-expressing bacteria of various stages of phagocytosis. Hemocyte surface, phagosomes and phagolysosomes are highlighted by staining for Hook (red, Krämer and Phistry, 1996) and Spinster (blue, Sweeney and Davis, 2002).
Hemocytes isolated by our procedure are mostly phagocytic active plasmatocytes, but also contain crystal cells that make up some 5% of the hemocyte pool (Tepass et al., 1994). Upon activation crystal cells secrete prophenoloxidase which is activated by proteolysis. Subsequently, the activated phenoloxidase initiates a melanization reaction that interferes with imaging of bacterial degradation products (Neyen et al., 2014). Therefore, after all the larvae have been ripped open and subsequently removed from the media, it is critically important to prevent the Schneider’s Drosophila medium from turning black. The melanization process will start around 10 min after the larval dissecting begins and will proceed with hemocytes cannibalizing other hemolymph cells (i.e., crystal cells). Other researchers (Tirouvanziam et al., 2004) added protease inhibitors to the isolation medium to suppress the protease cascade that initiates melanization. We found, however, that our approach was sufficient to minimize melanization, while avoiding any potential effect of the protease inhibitor on phagosome maturation and bacterial degradation.

Recipes

10x PBS stock solution
80 g NaCl
2 g KCl
26.8 g Na₂HPO₄·7H₂O
2.4 g KH₂PO₄
Dissolve in 800 ml ddH₂O
Adjust pH with HCl to 7.4
Add ddH₂O to 1 L and mix well
8% paraformaldehyde stock solution

Heat 200 ml dH₂O to 55 °C (but not warmer!)
Add 2 drops 50% w/w NaOH
Add 20 g paraformaldehyde
Stir continuously until clear
Vacuum filter through with 0.22 µm filter
Add ddH₂O to adjust final volume to 250 ml
Aliquot 2 ml into 15 ml Falcon tubes
store at -80 °C

Fixative: 4% paraformaldehyde in PBS

Thaw a 2 ml aliquot of 8% paraformaldehyde
Add 1.6 ml dH₂O
Add 400 µl 10x PBS stock solution
Mix well

Acknowledgments

The work herein was supported by NIH Grants EY010199, EY021922. This protocol has been adapted and modified from our previous published work (Akbar et al., 2011; Akbar et al., 2016). The authors of this work declare no conflicts of interest.

References

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