Materials and Reagents

1. Pipet tip for gel loading (Vertex-GL 200 μl gel-loading tip) (SSIbio, catalog number: 4837-S0S )
   
2. Parafilm
   
3. 1 ml sterile syringe (without needle) (TERUMO, catalog number: SS-01T )
   
4. Mice (C57BL/6, BALB/c etc.)
   Note: For training, bigger mice (aged male mice) are better.
   
5. Alhydrogel (InvivoGen, catalog number: vac-alu-250 ) as particulate for instillation
   Note: Alhydrogel (alum) is suspended in dH₂O. For instillation of alum into the lungs, buffer exchange is required. Centrifuge a suspension of alum in a microtube at 2,000 x g for 2 min. Discard supernatant (H₂O) and add an equal volume of saline or PBS. Mix well and centrifuge again. Repeat this procedure five times to exchange H₂O to saline or PBS. Finally, adjust the concentration of alum to 2 mg/ml in saline and use for instillation.
   
6. Anesthetic (ketamine/xylazine mixture)
   Note: 10 ml of Ketalar (Ketamine, 50 mg/ml, Daiichi Sankyo Co. Ltd., Tokyo, Japan) is mixed with 2.2 ml of Selactar (Xylazine, 20 mg/ml, Bayer HealthCare Ltd., Tokyo, Japan). Anesthetize mice with 50 to 75 μl of ketamine/xylazine mixture by s.c. injection into the back.
   

Equipment

1. Ear pick earwax remover with light (Japan Smile Kids)
   Note: Before use, silicone rubber at the tip of ear pick will be removed (Figure 1).
   
2. Stainless steel micro spatula (Figure 1) (ASONE, catalog number: 9-891-02 )
   Note: This is used as a tongue depressor, so small size is better.
   
   
   Figure 1. Ear puck with light and spatula
   
   
3. Platform for instillation into the lungs. As shown in Figure 2, stretch a string across the wooden (cork) board (approximate size: 20 x 30 cm)
   
   
   Figure 2. Platform for instillation into the lungs
   
   
4. Injector for instillation (Figure 3)
   
5. FACS analyzer
   
   
   Figure 3. Injector for instillation. A. Fix a thin pipet tip to the ear pick by Parafilm. B. Attach a syringe to the pipet tip. C. If needed, cut the top of pipet tip to adjust for syringe insertion. D. Cut the edge of the pipet tip (arrow) for injection.
   

Procedure