Preparation of single hemocytes in suspension

Isolation of blood cells or hemocytes from Drosophila late L3 larvae for scRNA-seq
 

Sudhir Tattikota, Ph.D.,

Perrimon Lab, Harvard Medical School

This protocol is adapted from various published literature together with suggestions from the inDrop and 10X single cell sequencing platforms. Further, this protocol aims to get sessile plus circulating hemocytes and may be modified a little if either of the two are preferred separately.

Things to have before beginning the entire protocol:

1. Precisely timed larvae 120 AEL or late L3
2. Ice bucket with ice
3. 9-well chambered glass plate (ultra clean)
4. Glass beads, non-sterile (Cat# G9268-100G, Sigma)
5. Optiprep gradient solution (Cat# AXS-1114542, Axis-Shield)
6. Cold centrifuge (with swinging bucket rotor) set to 4 degree
7. Thin #5 tweezers (two)

Timeline:

Expect the following time in minutes before you reach the 10X for encapsulation.

1. Larvae collection and clean-up: ~30min
2. Transferring to eppendorf tubes and vortexing: ~3min
3. Dissection/Bleeding: ~15min
4. Optiprep spin including “no brake” time: ~30min
5. Cell recovery and counting: ~10-15min

So total time would be around 90min ~ 2hr for two samples with at least 100 larvae each sample.

Prepare Optiprep solution beforehand. See below;

The best concentration that worked in my case was 1.09 g/ml. To achieve this, simply mix Optiprep and PBS at a ratio of 0.27 (Optiprep): 0.73 (PBS).

That is for 2ml just mix 540ul of optiprep in 1460ul of PBS. Mix thoroughly in a 15ml falcon tube and place on ice.

Stepwise protocol to get high quality hemocytes from 3rd instar larvae:

1. Collect precisely timed ~100-200 larvae for each condition in PBS.
2. Wash them briefly and load all of them in 2ml Eppendorf tubes containing ~500 ml of glass beads and ~200ul PBS/water.
3. Vortex the tubes containing larvae for about 2 min at high speed
4. Decant the larvae with beads (you may have to use water to force out the larvae and beads from the tubes) in a petri dish and collect the larvae and subsequently transfer to a 9-well plate containing 200 ml PBS in one of the cells
5. Next, transfer the larvae in another well containing 100 ml of “ice cold” PBS or Schneider's media or PBS with 0.5% BSA (sterile filtered)
6. Don’t be shy to collect a few glass beads as well along the way as they will disallow any tissue chunks/ruptured larvae from clogging the pipette tips during the cell collection step.
7. Gently nick open the posterior end of every larva using tweezers and low all the larvae to bleed at least for a minute (Petraki et al., 2015)
8. Pipette out the entire cells in PBS/media using a P200 pipette and transfer to a clean Eppendorf tube by filtration using a 100 mm cell strainer to get rid of chunks and debris
9. Next, gently overlay the filtered cell suspension onto 2ml of 1.09 g/ml Optiprep Gradient (as prepared in 15 ml falcon tube) that is sitting on ice
10. Proceed to the next condition of larvae (if applicable), get the cells (steps 1-8), and overlay onto another Optiprep gradient. Ideally, I would collect the two conditions of larvae sequentially and place the larvae from the two conditions in ice cold PBS in two separate wells and bleed them one condition after the other and collect the cells (Make sure all larvae receive at least a minute or two for the cells to bleed out)
11. Spin the Optiprep with cells at 1800 - 2000 rpm at 4 degree in a swingling bucket rotor for about 20 min with “no brake” setting.
12. Carefully collect the cells over the gradient. Here I would first collect the top 100 ml that you loaded and transfer to a clean Eppendorf tube. I would also collect additional 100 ml from top and transfer to another clean Eppendorf tube. Count cells from both tubes and see which ones have more cells. Take the one with more cells. Sometimes the cells slip to the next 100 ml region over the Optiprep.
13. Count the cells using Haemocytometer (Neubar chamber) or BioRad automated cell counter.
14. 10X platform recommends 1000 cells/ml. This can be achieved by additional spin and resuspending the cells in a lesser volume of PBS with 0.5% BSA. Ideal numbers in my opinion: 700~1000 cells per ml.
15. Once the encapsulation is successfully done, follow the exact amplification and library prep steps designed by the 10X. Exactly the same and you will get decent quality of library as recommended by the 10X.

ALTERNATIVE Quick protocol:

Alternatively, the Optiprep step can be avoided if the sample preparation is clean with far less dead material, dead cells and cell clumps. Below is a quick alternative protocol without the Optiprep step. Importantly, this method may also help in obtaining higher number of Hemocytes.

1. Follow steps 1-8 of the stepwise protocol above
2. After bleeding, filtering the cell suspension through 100 mm cell-strainer, transfer the cell suspension to clean Eppendorf tubes
3. Centrifuge at 1000g or ~3000 rpm at 4 degrees for 5 min
4. Remove the supernatant and resuspend in 100 ml of PBS-0.5% BSA solution.
5. Repeat steps 3-4 for a total of two washes
6. Count the cells using hemocytometer
7. Recommended cell numbers for 10X: 700~1000 cells per ml. This is a sweet spot to achieve 10K cell recovery after encapsulation.

Key References:

1. Petraki et al., JoVE, 2015 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692709/
2. Notes on Optiprep http://www.axis-shield-density-gradient-media.com/C13.pdf
3. For inDrop platforms see “Helpful information” section that is available on https://iccb.med.harvard.edu/single-cell-core

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