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Nystatin and subunit vaccine injection in mice

BT Beth Ann Jiron Tamburini

Published: Jul 14, 2023 Views: 950

Original research article

The authors used this protocol in:

https://doi.org/10.7554/eLife.62781

Apr 12, 2021

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  1. Nystatin (Sigma N4014) was resuspended in DMSO to a concentration of 10 mg/mL. Nystatin was then aliquoted and stored at -20 C. When possible nystatin was made fresh. 
  2. Mice were injected using a tuberculin syringe with 50 μL of 10 mg/mL nystatin in the footpad.  Footpad injection was performed under anesthesia (isofluorane) 
  3. Let mice rest for 1 hour. 
  4. After 1 hour mice were injected in the same footpad with 5 ug of ova conjugated to Alexa 488, 2.5 ug of anti-CD40 (BioXcell, Clone: FGK4.5, stock at 2 mg/mL) and 2.5 ug of polyI:C (Invivogen, stock at 1 mg/mL) at a final volume of 50 uL per footpad.
  5. Mice were euthanized 24 hours later with CO2 and popliteal lymph nodes were dissected and placed in a 6 well tissue culture dish in 2 mL of Click’s EHAA media (Fujifilm). 
  6. Lymph nodes were minced with a 22-gauge needle to mechanically break up the tissue. 
  7. Lymph node tissue was digested by adding 0.25 mg of liberase dispase low (DL) (Sigma-Aldrich Cat. No. 5466202001) and DNAse (Worthington Biochemical Cat. No. LS002145) to the 2ml of Click’s EHAA media in a plate for 1 hr at 37 degrees C. Tissue was pipetted every 15 minutes to physically separate the tissue. By the end of the digestion period tissue should be broken up.
  8. Following digestion, to further process into a single cell suspension, cells were passed through a 100-micron screen and pushed through with the back of a 1ml syringe.  Screen was washed with 5 mM EDTA + 2-5% FBS in Click’s EHAA media to stop the digestion. 
  9. Cells were then spun down at 1400 RPM for 5 min. 
  10. You should see a small pellet of cells. Remove media from pelleted cells. 
  11. Cells were washed with FACs buffer (0.1% bovine serum albumin (BSA), 1x hank’s buffered saline solution, 1mM ethylene diamine tetra actetic acid (EDTA) and 0.05% sodium azide) 1x, spinning at 1400RPM for 5 minutes.
  12. Cells were stained with CD45 brilliant violet 510 (Biolegend clone 30F11, 1:300), PDPN APC (Biolegend clone 8.1.1, 1:200), CD31 PercP Cy5.5 (Biolegend clone 390, 1:200), and PD-L1 pacific blue (Biolegend clone 10F.9G2, 1:200) in 10% 24G2 (Fc block) for 30 minutes at 4 degrees C. 
  13. After staining, wash twice with FACS buffer at 1400 RPM for 5 minutes and run on the cytometer.
Copyright: © 2023 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).

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