All tissues were acquired after ethical clearance from the institute ethics committees of Indian Institute of Technology Jodhpur and All India Institute of Medical Sciences (AIIMS) Jodhpur.

1.Tissue acquisition and processing (Day 0)

1. Collect the tissue in a 50 mL tube containing 10 mL of ice cold artificial cerebrospinal fluid (aCSF) (2 mM CaCl₂∙2H₂O, 10 mM glucose, 3 mM KCl, 26 mM NaHCO₃, 2.5 mM NaH₂PO₄, 1 mM MgCl₂∙6H₂O, 202 mM sucrose)²⁰. Ensure that the tube is kept on ice if the tissue needs to be transferred to a different location.
   NOTE: Prepare aCSF in autoclaved distilled water. Filter it with 0.22 µm syringe filter in the laminar hood. This can be stored for 1 month at 4 °C.
2. Wipe the collection tube carefully with 70% alcohol and transfer to an aseptic laminar air flow chamber.
3. Discard aCSF carefully and weigh tissue in an aseptic condition. Tissue weight is essential to calculate the volume of trypsin-EDTA needed for subsequent steps.
4. Keep the tissue in fresh warm aCSF at 37 °C for 5 minutes. This step is critical to avoid cell death.
5. Discard aCSF and wash tissue once with 1x PBS (phosphate buffered saline) at 37 °C. Ensure all blood is washed away with repeated PBS washes (as needed).
6. Incubate the tissue in warm PBS, at 37 °C, for 5 min.
7. Discard PBS carefully and transfer tissue to a sterilized Petri dish. PBS may be removed with a pipette. This will prevent any loss of tissue.
8. Dice the tissue into small (at least 1 mm³) pieces using a sterile scalpel. Finely diced tissue provides higher tissue surface area for tissue dissociation by trypsin-EDTA ensuring higher yield.
9. Transfer the diced tissue to a 50 mL tube containing 10 mL/g tissue of 0.25% trypsin-EDTA and mix by pipetting through a 10 mL serological pipette. Add 2 mL of trypsin/EDTA to a Petri dish and wash the plate thoroughly with the help of pipette. Add this trypsin back to the falcon tube. This minimizes loss of tissue and cells while dicing.
10. Incubate the tube on a shaker for 30 minutes at 37 °C at 250 rpm. This step increases the dissociation of cells from tissue.
11. At the end of the incubation, add 10 mL of neutralizing medium (50% DMEM/50% F12 with glutamine, 1% penicillin-streptomycin, 10% FBS) to neutralize trypsin. Mix with a 10 mL serological pipette. The amount of neutralizing media added should be equal to the amount of trypsin used.
12. Centrifuge the tube at 2,000 x g at 4 °C for 10 minutes.
13. Discard the supernatant and re-suspend the pellet in 1 mL of culture medium (50% DMEM/50% F12 with glutamine, 1% penicillin-streptomycin, 20% L929 supernatant, 10% FBS).
    NOTE: L929 cells are culture in DMEM (DMEM with glutamine, 1% penicillin-streptomycin, 10% FBS). ATCC recommended culture method should be followed for cell culture. Supernatant must be collected from the culture flask which is at least 75% confluent. It can be collected in bulk and stored at -80 °C to prevent degradation of growth factors. It is recommended to add L929 supernatant separately in flasks instead of adding to the stock culture medium.
14. Plate the cells in a T-25 flask, suited for adherent cells, and add 4 mL of additional culture medium. Incubate the flask at 37 °C with 5% CO₂. Carefully shake the flask to homogenously disperse the tissue. Avoid bringing the media to the neck of the flask, while shaking, as this may increase the chances of contamination.

2.Cell culture (Day 2)

1. Collect the media from the T-25 culture flask prepared on day 0 in three 1.5 mL centrifuge tubes by collecting equal volume of media in each tube. Wash the flask once with 1 x PBS. Shake the flask gently to remove any remnant tissue fragments left. Avoid harsh shaking of the flask as any remnant fragments will not adversely affect the culture. Add 5 mL fresh culture media to the flask.
2. Centrifuge the collected media at 1,466 x g (4000 rpm) at 4 °C for 4 minutes.
3. Discard the supernatant from each tube and add 1 mL of culture medium to one of the tubes. Mix thoroughly with pipette. Serially add the mixed media with cells to other tubes. Mix thoroughly with pipette and pool the cells in one tube.
4. Plate the cells in a separate T-25 flask, suited for adherent cells. Add 4 mL of culture medium and incubate the flask at 37 °C with 5% CO₂.

3.Cell culture (Day 4)

1. Discard the media from both flasks and add fresh 5 mL of culture media to the flask.
2. Incubate the flask at 37 °C with 5% CO₂ for 2 days.

4. Cell Culture (Day 6)

1. Cells will be ready for further experiments.