TITLE: Labeling of host immune cells with magnetic nanoparticles
AUTHORS: Eric J. Koch, Jamie S Foster

[DESCRIPTION]
This protocol describes a technique to label the innate immune cells of a host squid, known as hemocytes, with commercially available magnetic nanoparticles (i.e., magdots). The goal of this technique is to label the hemocytes so that they could be reintroduced into the host animal and be tracked with downstream applications, such as fluorescence and magnetic particle imaging, or manipulated with techniques such as magnetic tweezers. The labeling technique can be applied to any cell type with phagocytic capabilities.




[STEPS]
SECTION: Preparation of target immune cells - squid hemocytes
1. Collect sample of target immune cells. Here, hemocytes extracted from the bobtail squid Euprymna scolopes were used.
SECTION: Preparation of target immune cells - squid hemocytes
2. Using an insulin 1 cc syringe (30 gauge needle) collect hemolymph from the cephalic artery in the bobtail squid as visualized in Collins and Nyholm, 2010. Typically, from an adult animal 200 µL can collected with a single bleeding event. Place the hemolymph in a 1.5 mL centrifuge tube.
SECTION: Preparation of target immune cells - squid hemocytes
3. Resuspend the hemocytes by vigorously pipetting the hemolymph 5 to 10 times. Be sure to break up any clumps of cells. 
SECTION: Preparation of target immune cells - squid hemocytes
4. Distribute the hemolymph into the desired number of 1.5 mL centrifuge tubes based on the experimental design (e.g., four tubes for four conditions).
SECTION: Preparation of target immune cells - squid hemocytes
5. Centrifuge the hemocytes at 2000 RCF, 4°C for 15 minutes to pellet the hemocytes.
SECTION: Preparation of Magnetic nanoparticles (i.e., magdots)
6. For this protocol, polyethylene glycol-coated MagDots that fluoresce at 610 nm from Core Quantum Technologies (https://www.corequantum.com/) was used. Many other types of particles are available as well as other wavelengths and coatings. Note the concentration of the commercial product will vary between purchases. The goal is to have the final concentration of the magdots to be 100 µg/mL, therefore, adjust the volumes accordingly.
SECTION: Preparation of Magnetic nanoparticles (i.e., magdots)
8. While the hemocytes are being pelleted, remove the total amount of magdots needed for all conditions and experimental treatments and place into a 1.5 mL centrifuge tube. Centrifuge the magdots at 10,000 RCF (or maximum speed) to pellet the magnetic particles and remove the supernatant containing the stabilization buffer that came with the particles. Note: you will likely have to leave a few µL of buffer behind (e.g. between 10 and 20 µL).
SECTION: Preparation of Magnetic nanoparticles (i.e., magdots)
9. Resuspend the magdots in the total amount of buffer necessary for all samples. In this case,  80 µL of Squid Ringer's solution (see materials for recipe; Nyholm et al., 2009) was added per sample (e.g. 400 µL of Squid Ringer’s would be required for five samples).
SECTION: Preparation of Magnetic nanoparticles (i.e., magdots)
10. Break up the magdot pellet with either a bath sonicator for 2 min or by vigorously pipetting until the pellet is dispersed.
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
11. Immediately after the hemocytes have been pelleted, remove cell-free hemolymph leaving only 20 µL (or 20% of the incubation volume if you are not using 100 µL).
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
12. Add the 80 µL of resuspended magdots in Squid Ringer's solution to each of the prepared tubes containing 20 µL of hemocytes for a final volume of 100 µL. Note the magdot final concentration should be 100 µg/mL.
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
13. For a negative control, add 100 µL of Squid Ringer's solution with no magdots added.
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
14. Incubate the hemocytes with the magdots overnight (between 12 - 24 h) in the dark at room temperature. 
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
15. Following the incubation, resuspend the hemocytes and magdots by pipetting vigorously several times.
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
16. Immediately centrifuge the samples at 500 - 1000 RCF for 15 min to pellet the cells but keeping the free, unincorporated magdots suspended.
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
17. Gently remove the supernatant containing the magdots and discard.
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
18. Rinse the now labeled hemocytes by suspending in 100 - 200 µL of Squid Ringer's solution and centrifuge for 500 - 1000 RCF for 15 min.
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
19. Repeat the rinsing process 3 to 5 times until there are no magdots visible in the supernatant.
SECTION: Co-incubation of Magnetic nanoparticles and immune cells
20. The labeled hemocytes are now ready for visualization or downstream application.
SECTION: Preparation of Magnetic nanoparticles (i.e., magdots)
7. Pipette 5 µL of magdots per sample for a final concentration of 10 µg/ 100 µL.
A typical labeling experiment would have 10 replicate hemocyte samples, therefore on average 50 µL of magdots are used per experiment.