Protocols

Collection, Processing, and
Culture of Schwann and Schwannoma Cells From Surgical Samples

Landegger LD, Sagers JE, Dilwali S, Fujita T, Sahin MI, Stankovic KM. A Unified Methodological Framework for Vestibular Schwannoma Research. JoVE. 2017;(124):55827. doi:10.3791/55827-v

Cochlear Explant Microdissection, Culture and Immunostaining

Landegger LD, Dilwali S, Stankovic KM. Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research. JoVE. 2017;(124):55704. doi:10.3791/55704-v

A Cerebellopontine Mouse Model for Vestibular Schwannoma

Chen J, Landegger LD, Sun Y, Ren J, Maimon N, Wu L, Ng MR, Chen JW, Zhang N, Zhao Y, Gao X, Fujita T, Roberge S, Huang P, Jain RK, Plotkin SR, Stankovic KM, Xu L. A cerebellopontine angle mouse model for the investigation of tumor biology, hearing, and neurological function in NF2-related vestibular schwannoma. Nat Protoc. 2019;14(2):541-555. doi:10.1007/978-1-0716-0856-2_9

Plasma Extraction

Step 1:
Withdraw blood into a BD Vacutainer® PPT™ Plasma Preparation Tube (EDTA).

Step 2:
Immediately and gently invert the tube 8 - 10 times and transport the sample on ice to the lab for blood processing.

Step 3:
Keep the tubes on ice or refrigerated and isolate plasma within 2 hours of the blood draw.

Step 4:
Pre-cool the centrifuge (4 °C).

Step 5:
Prepare the Necessary Materials

  • Blood tube
  • Filter 0.2 µm
  • Needle and Syringe 3mL
  • About 3 cryovials (1.5 ml) per blood tube
  • 2 Falcon tubes 15 mL
  • Automatic Pipette 1 mL and tips

Step 6:
Centrifuge BD PPT™ Tube in a balanced, swing-out rotor type centrifuge (4 °C) at 2000xg for 10 minutes.

Step 7:
Obtain a tube with an undiluted plasma sample. The tube contains 3 different layers:

  • top – plasma
  • middle– buffy coat, platelets
  • bottom– red blood cells)

Step 8:
Remove Plasma from Red Blood Cells

  • Remove the Hemogard Closure (the colored top on the blood tube) from the test tube and open a 15 mL Falcon tube.
  • With a prepared syringe, remove the plasma from the blood collection tube and transfer the plasma to a 15 mL Falcon tube.
  • Avoid disturbing the lower layers: the buffy coat, platelets, and red blood cells.

Step 9:
Centrifuge the Collected Plasma

Centrifuge the tube with plasma at 2000xg for 5 minutes (4°C).

You should be able to see the separated plasma from the red blood cells. Place the tube back on the ice.

Step 10:
Build up Syringe/Filter/Collection system

  • Syringe 3mL (BD™ Luer Slip Tip Syringe sterile, single use, 3 mL, 309656, BD)
  • Millex-GP Syringe Filter Unit, 0.22 µm (SLGPM33RS, Millipore Sigma)
  • 15 mL falcon tube
     

Step 11:
To filter the plasma, transfer the supernatant to the: Syringe/Filter/Collection system using 1 ml tips and pipette.

Do not take the residue.
 

Step 12:
Aliquot 1.0 mL of filtered plasma into cryopreservation vials.

Label vials with sample ID, date, “Plasma Filtered,” and initials.

Step 13:
Transfer on ice the labeled plasma vials and the blood tube containing the buffy coat, platelets, and red blood cells, and then to a -80 º C freezer for storage.