TITLE: Sinai SCENT TMC - 5x5 Project Mouse Harvesting
AUTHORS: Sojin Kim

[DESCRIPTION]
We propose a total of 10 C57BL/6 mice (5 males and 5 females) to be studied for each study group or cellular senescence inducer intervention.

[STEPS]
SECTION: Experimental Design
1. Males and females are grouped and housed in standard cages, provided LabDiet 5K0G and acidified water ad libitum. Euthanasia is performed by cervical dislocation (CMQ93-26).

Control: Tissues collected at 32 weeks.
Doxorubicin: IP injection (5 mg/kg) at 26 weeks on days 0 and 10, with tissue collection at 32 weeks.
Palbociclib: Daily oral gavage (150 mg/kg) for 10 days starting at 26 weeks, with tissue collection at 32 weeks.
High-Fat Diet: From 24 weeks, animals receive a 45% fat diet until tissue collection at 32 weeks.
Aging Controls: Tissues collected at 24 months.


SECTION: Lung Tissue Collection
3. Preparation: After euthanasia, apply 70% ethanol to the throat to wet the fur.
SECTION: Lung Tissue Collection
4. Incision: Lift the skin and make a vertical cut on the throat to expose the salivary glands. Use forceps to expose the trachea.
SECTION: Lung Tissue Collection
5. Tracheal Access: Make a horizontal incision between the second and third tracheal rings. Insert a 19G cannula and secure it with a suture thread.
SECTION: BAL Fluid (BALF) and BAL cell separation
14. Centrifuge BAL sample: 3,000 rpm, 10 min, 4°C
SECTION: BAL Fluid (BALF) and BAL cell separation
15. Transfer the supernatant (=BALF) to a new E-tube (label: BALF) → dry ice or liquid nitrogen → -80'C
SECTION: Bronchoalveolar Lavage (BAL) Collection
11. Slowly inject 0.8 mL of ice-cold PBS into the lungs (speed: 0.1 mL/sec), pause for 2 – 3 sec, and then slowly retrieve the BAL. Record the collected volume of BAL and transfer BAL to theE-tube (label: BAL cells) on ice. Repeat Steps with another 0.8 mL of ice-cold PBS. Save the BAL in one tube/sample.
SECTION: Bronchoalveolar Lavage (BAL) Collection
12. Save the collected BAL on the ice during harvest and then continue for the “BALF and BAL cell separation”
procedure
SECTION: Lung Tissue Collection
6. Optional: Go to "Bronchoalveolar Lavage Collection"
SECTION: Lung Tissue Collection
8. Lung Isolation: Carefully remove the lung with the trachea and cannula intact.
SECTION: Lung Tissue Collection
9. Tissue Processing:
Right Lobe: Isolate, freeze in liquid nitrogen, and store at -80°C.
Left Lobe: Fix in 10% formalin overnight, transfer to 70% ethanol, and prepare for histology.
SECTION: BAL Fluid (BALF) and BAL cell separation
16. CRITICAL: Cell pellet + 1 mL room temperature RBC lysis buffer → immediately vortex for 3 seconds →
Incubation: 5 min, ice. Incubation time is critical.
SECTION: BAL Fluid (BALF) and BAL cell separation
17. Centrifuge: 3,000 rpm, 10 min, 4°C
SECTION: BAL Fluid (BALF) and BAL cell separation
18. Discard supernatant using a pipette. Do not disturb the pellet.
SECTION: BAL Fluid (BALF) and BAL cell separation
19. If you want to count the WBC, proceed to step 5. Or you can save BAL cell → dry ice or liquid nitrogen →
-80'C
SECTION: BAL Fluid (BALF) and BAL cell separation
20. Optional: Cell pellet + 500 uL of ice-cold PBS. Make the single-cell suspension by pipetting. CRITICAL Un-even cell suspension affects cell number as well as the quality of the Cytospin
SECTION: Blood Collection and Serum Separation
22. Open the abdominal cavity. Remove all abdominal organs to reveal the inferior vena cava, which is located in the middle of the back of the body.
SECTION: Lung Tissue Collection
7. Optional: Go to "Blood Collection and Serum Separation"
SECTION: Blood Collection and Serum Separation
23. Insert a 26G syringe into the inferior vena cava and collect the blood slowly.
SECTION: Blood Collection and Serum Separation
24. Transfer the collected blood to the e-tube. CRITICAL do not drop the blood. gently transfer the blood from
the syringe to E-tube unless RBC is destroyed and affects the colorimetric analysis.
SECTION: Blood Collection and Serum Separation
25. Leave the tube at room temperature for 30 min. Do not close the lid
SECTION: Blood Collection and Serum Separation
26. Close the lid and centrifuge: 1,000 g, 10 min, 4°C
SECTION: Blood Collection and Serum Separation
27. Collect the supernatant (=serum) carefully and transfer it to a new e-tube. Saved it at -80'C
SECTION: Lung Tissue Collection
2. Materials: 26G needle, 1 mL syringe, balance, surgical board, 70% EtOH, scissors, 2 forceps, suture thread, 19G cannula, E-tube (1 per sample), normal formalin, tools for lung inflation, histology container with 10 mL of normal formalin (1 per sample)
SECTION: Bronchoalveolar Lavage (BAL) Collection
10. Materials: 26G-syringe, 70% ethanol spray, surgery board with needles/pins, 2 forceps, 1 scissors, 4.0-suture string, 19G-cannula (brown color), PBS, 1 mL syringe, e-tube, notebook/paper, pens
SECTION: BAL Fluid (BALF) and BAL cell separation
13. Materials: 1 mL pipette, tips, e-tubes, marker pen, dry ice, waste bin, e-tube rack, timer, PBS (keep on ice), ice bucket, RBC lysis buffer
SECTION: Blood Collection and Serum Separation
21. Materials: 26G-syringe, surgery board with needles/pins, 1 forceps, 1 scissors, 23G-syringe, e-tube