Ex vivo larval hemocyte phagocytosis assays

Overview

This assay quantifies phagocytic activity of Drosophila larval hemocytes using fluorescent bacterial bioparticles and flow cytometry. Hemocytes are identified/gated using Hml-Gal4, UAS-GFP, and phagocytosis is assessed by uptake of Alexa Fluor 488–labeled bioparticles.

Step 1: Definition of Hemocyte Gating Using Hml-Gal4, UAS-GFP

This step is performed periodically to establish robust hemocyte gates and instrument settings on the CytoFLEX using a Hml-Gal4, UAS-GFP line, enabling subsequent analysis of non-GFP genotypes using the same template.

Materials

• Hml-Gal4, UAS-GFP third-instar larvae
• Schneider’s insect medium (pre-chilled on ice)
• Protein Lo-Bind Eppendorf tubes
• Glass slide and fine forceps
• CytoFLEX flow cytometer (488 nm laser for GFP/Alexa 488 detection)
• Fluorescent bacterial bioparticles (Alexa Fluor 488, Thermo Fisher) for setting the bioparticle signal.

Hemocyte Collection (Template Setup)

• Collect 5 third-instar Hml-Gal4, UAS-GFP larvae in a microcentrifuge tube.
• Briefly vortex (5–10 s) to remove external debris.
• Surface-sterilize the larvae (e.g., quick rinse in 70% ethanol followed by Schneider’s medium).
• Place the larvae on a clean glass slide and add 150 µL ice-cold Schneider’s medium.
• Gently tear the larvae with fine forceps to release hemolymph while keeping the fat body and gut largely intact.
• Carefully remove visible tissues and debris from the drop.
• Transfer the hemolymph-containing medium into a Protein Lo-Bind tube and keep the sample on ice until acquisition (within approximately 15–20 minutes in total).

Hemocyte Gating Strategy (Hml-Gal4, UAS-GFP)

Hemocytes are identified and gated based on FSC/SSC properties and GFP signal from Hml-Gal4, UAS-GFP larvae.

Primary FSC vs SSC Gate (All Events)

• Plot FSC-A versus SSC-A on a logarithmic scale.
• Draw a broad gate that encompasses the main cell cloud while excluding obvious debris and large aggregates.
• This serves as the initial “all events” gate and will be refined in subsequent steps.

Histogram of GFP Signal

• From the primary FSC/SSC gate, generate a GFP (e.g., FITC channel) histogram of all events.
• Identify the GFP-positive population using Hml-Gal4, UAS-GFP larvae and a non-GFP control to define the GFP threshold.

Definition of GFP+ Hemocyte Gate (P1) on FSC vs SSC

• In the FSC-A versus SSC-A plot, display GFP intensity (e.g., as a color overlay or biaxial plot) and identify the region where events exhibit clear GFP signal.
• Draw a refined gate around this GFP-positive cell cloud; this is the P1 hemocyte population, representing Hml>GFP+ hemocytes in FSC/SSC space.

Singlet Gate Using FSC-A vs FSC-H

• Using P1 as the parent population, plot FSC-A versus FSC-H.
• Single cells form a tight diagonal distribution (approximately 45°), whereas doublets and aggregates deviate from this line.
• Draw a gate around this diagonal single-cell population; this is the singlet hemocyte gate (often labeled P2).

Confirmation of GFP in Singlets (Optional)

• From the singlet (P2) gate, generate a GFP histogram to confirm that the gated population remains strongly GFP-positive and that GFP-negative/background events are excluded.

Saving the Gating Strategy

• Save the complete gating hierarchy (primary FSC/SSC gate, P1 GFP+ hemocytes, and P2 FSC-A vs FSC-H singlets) as a template in the cytometer software.
• This template defines the hemocyte population and will be reused for subsequent phagocytosis experiments and for non-GFP genotypes by applying the same FSC/SSC and singlet gates.

Flow Cytometer Setup and Template Creation

• Run a tube containing fluorescent bacterial bioparticles diluted in Schneider’s medium (no cells).
• Use the 488 nm laser and record the Alexa 488 channel.
• Adjust the detector gain so that the bioparticle fluorescence falls within an appropriate intensity range (e.g., mid-log scale) to provide adequate dynamic range for subsequent samples.
• Save these detector/gain settings and plot configurations (FSC vs SSC, dot plots, Alexa 488 histograms) as part of the same template in the CytoFLEX software (e.g., “Hemocyte_phago_template”).

Step 2: Ex Vivo Phagocytosis Assay with Non-Hml-Gal4, UAS-GFP Genotypes

In this step, the previously defined template (including FSC/SSC and singlet gates, as well as the phagocytic gate) is applied to other genotypes that do not carry Hml-Gal4, UAS-GFP.

Materials

• Test genotypes: third-instar larvae lacking Hml-Gal4, UAS-GFP
• Schneider’s insect medium (ice-cold)
• Fluorescent bacterial bioparticles (e.g., Alexa Fluor 488–labeled, Thermo Fisher)
• Protein Lo-Bind Eppendorf tubes
• CytoFLEX flow cytometer configured identically to Step 1 and using the saved template.

Hemocyte Collection for Test Genotypes

• Collect 5 third-instar larvae of the test genotype and surface-sterilize as described in Step 1.
• Place the larvae on a slide with 130 µL ice-cold Schneider’s medium.
• Tear the larvae to release hemolymph, avoiding excessive disruption of internal organs.
• Remove fat body and gut debris from the drop.
• Transfer the hemolymph-containing medium to a Protein Lo-Bind tube.
• Aim to add bioparticles within 1 minute of bleeding to minimize variability between samples.

Ex Vivo Phagocytosis Reaction

• Add approximately the desired number of fluorescent bacterial bioparticles (after vortexing the stock) to the hemocyte suspension, and tap gently on the side of the tube to mix.
• Incubate at room temperature for 1 hour to allow phagocytosis.
• After 1 hour, place the tube on ice to stop further uptake.
• Keep the samples on ice and acquire them on the CytoFLEX within 30 minutes of stopping the reaction.

Flow Cytometry Acquisition of Test Genotypes

• Load the previously saved “Hemocyte_phago_template” in the CytoFLEX software to import:
  • Detector and gain settings (FSC, SSC, Alexa 488).
  • The gating tree (P1: main cells; P2: singlets; Phago+ gate).
• Run a medium-only tube to confirm that the baseline signal matches the conditions used in Step 1.
• Acquire the test genotype sample:
  • Use the same flow rate (40 µL/min) and stopping condition (60 µL) as in the template setup.
  • Inspect the FSC vs SSC plot to confirm that the main cell population falls within the P1 gate defined using Hml>GFP larvae. If the population is slightly shifted due to genetic background, adjust only minimally and maintain consistency across all test genotypes.

Gating in the Absence of GFP

Because the test genotypes lack Hml-Gal4, UAS-GFP, the GFP gate is not used; however, P1 and P2 remain valid based on FSC/SSC morphology and singlet discrimination.

• Use the P1 gate (main hemocytes) defined in the template to exclude small debris and large aggregates.
• Apply the singlet gate (FSC-A vs FSC-H; P2) within P1 so that only single hemocytes are analyzed.
• Use the pre-defined Phago+ gate on the Alexa 488 channel (with the same fluorescence threshold as in Step 1) to distinguish bioparticle-positive from bioparticle-negative cells.

Data Output

For each sample (genotype), within the singlet P2 population:

• Determine the percentage of cells in the Alexa 488–positive (Phago+) gate.
• Determine the mean fluorescence intensity (MFI) of Alexa 488 within the Phago+ population.

Phagocytic Index (PI)

Define the phagocytic index (PI) as:

PI = f * MFI 
 

where is either the fraction (0–1) or percentage (0–100) of Alexa 488–positive cells, and MFI is the mean fluorescence intensity of the Phago+ population. 
Use the same definition of consistently across experiments.

Practical Notes

• Recalibrate periodically: repeat Step 1 every few weeks or whenever the cytometer configuration is changed to ensure that FSC/SSC and phagocytosis gates remain appropriate.
• Maintain identical acquisition settings and gating for all genotypes within an experiment to facilitate reliable comparisons