Materials and reagents

Biological materials

1. RAW 264.7 cells (ATCC, catalog number: TIB-71)

2. .Leishmania (L.) amazonensis promastigotes (strain IFLA/BR/67/PH8) (Norma W. Andrews, University of Maryland, College Park, MD)

3. L. amazonensis-mNeon, expressing bright monomeric green fluorescent protein, mNeonGreen [9]

1. Dulbecco’s modified Eagle’s medium (DMEM), with 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate (Sigma-Aldrich, catalog number: D6429), store at 4 °C

2. Benchmark fetal bovine serum (FBS), heat inactivated (GeminiBio, catalog number: 100-106-500-HI), store at -20 °C

3. Penicillin/streptomycin solution (GeminiBio, catalog number: 400-109-100), store at -20 °C

4. Hygromycin B solution (50 mg/mL) (GeminiBio, catalog number: 400-123-020), store at 4 °C

5. Schneider’s Drosophila medium (Sigma-Aldrich, catalog number: S9895), store at 4 °C

6. Hemin (Sigma-Aldrich, catalog number: H9039), store at 4 °C

7. Sodium hydroxide solution, 10 M in water (Sigma-Aldrich, catalog number: 72068)

8. Phosphate buffered saline (PBS), pH 7.4, liquid, sterile-filtered, suitable for cell culture (Sigma-Aldrich, catalog number: 806552), store at 4 °C

9. Formaldehyde, 37% in water (Sigma-Aldrich, catalog number: F1635)

10. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number A7906), store at 4 °C

11. Triton X-100, molecular biology grade (Sigma-Aldrich, catalog number: T8787)

12. Mouse anti-p8 antibody, generated from hybridoma cell line generously donated from McMahon-Pratt laboratory, Yale University; available upon request, store at -20 °C

13. Anti-mouse IgG (H+L), F(ab')2 fragment, CF-488A-conjugate antibody produced in goat (Sigma-Aldrich, catalog number: SAB4600388), store at -20 °C

14. Anti-mouse IgG (H+L), highly cross-adsorbed, CF-568-conjugate antibody produced in donkey (Sigma-Aldrich, catalog number: SAB4600075), store at -20 °C

15. Hoechst 33342 solution, 20 mM (Thermo Fisher, catalog number: 62249), store at 4 °C

16. Alexa Fluor^TM 647 Phalloidin (Thermo Fisher, catalog number: A22287), store at -20 °C

1. Complete RAW 264.7 cell media (see Recipes)

2. 50 mM sodium hydroxide (see Recipes)

3. Hemin solution (see Recipes)

4. Complete promastigote media (see Recipes)

5. Complete amastigote media (see Recipes)

6. Fixing solution (see Recipes)

7. Blocking solution (see Recipes)

8. Permeabilization solution (see Recipes)

Recipes

1. Complete RAW 264.7 cell media, final pH 7.4 (4 °C storage)

   Reagent	Final concentration	Quantity
   DMEM	n/a	440 mL
   FBS	10%	50 mL
   Penicillin/streptomycin solution	1×	5 mL
   Hygromycin	1×	5 mL
   Total	n/a	500 mL




2. 50 mM sodium hydroxide

   Reagent	Final concentration	Quantity
   10 M sodium hydroxide	50 mM	0.5 mL
   Water	n/a	99.5 mL
   Total	n/a	100 mL




3. Hemin solution (4 °C storage)

   Reagent	Final concentration	Quantity
   Hemin	2.5 mg/mL	25 mg
   50 mM sodium hydroxide	n/a	10 mL
   Total	n/a	10 mL




4. Complete promastigote media, final pH 7.4 (4 °C storage)

   Reagent	Final concentration	Quantity
   Schneider’s Drosophila medium	n/a	835 mL
   FBS	15%	150 mL
   Penicillin/streptomycin solution	10%	10 mL
   Hygromycin	1×	5 mL
   Total	n/a	1000 mL




5. Complete amastigote media, final pH 5.4 (4 °C storage)

   Reagent	Final concentration	Quantity
   Schneider’s Drosophila medium	n/a	785 mL
   FBS	20%	200 mL
   Penicillin/streptomycin solution	1×	10 mL
   Hemin solution (Recipe 3)	0.5%	5 mL
   Total	n/a	1,000 mL

   Adjust final pH to 5.4.




6. Fixing solution (-20 °C storage)

   Reagent	Final concentration	Quantity
   Formaldehyde, 37% in water	4%	5 mL
   1× PBS	n/a	45 mL
   Total	n/a	50 mL




7. Blocking solution (4 °C storage)

   Reagent	Final concentration	Quantity
   BSA	5%	2.5 g
   1× PBS	n/a	50 mL
   Total	n/a	50 mL




8. Permeabilization solution

   Reagent	Final concentration	Quantity
   Triton X-100	5%	0.5 mL
   Blocking solution	n/a	9.5 mL
   Total	n/a	10 mL

Laboratory supplies

1. Sterile 15 mL polypropylene centrifuge tubes (any vendor)

2. Sterile 50 mL polypropylene centrifuge tubes (any vendor)

3. Nalgene rapid-flow sterile disposable filter units 1 L (Thermo Scientific, catalog number: 5670020)

4. Sterile nuclease-free filter tips (10, 200, and 1,000 μL) (any vendor)

5. Vented flask surface area 25 cm² (any vendor)

6. Sterile cell culture dish, 100 mm (Greiner Bio-One, catalog number: 664160)

7. Sterile cell scraper (Falcon, catalog number: 353089)

8. PhenoPlate 96-well, black, optically clear flat-bottom, tissue-culture treated microplate (PerkinElmer, catalog number: 6055300)

9. Disposable plastic serological pipettes, sterile, individually wrapped, 5 mL, 10 mL, 25 mL capacity (any vendor)

10. Hemocytometer (Hausser Scientific, catalog number: 3200)

11. Microcentrifuge 1.7 mL tubes (Corning, Axygen^®, catalog number: MCT-175-C-S)

12. Bleach (Clorox)

13. 50 mL reagent reservoir (Corning, catalog number: 4870)

14. Poseidon^TM 12 channel pipettor P200, adjustable volume, 20–200 µL (Genesee Scientific, catalog number: 33-GSC-12P200)

15. Plastic container for waste collection, 1 L size (any vendor)

Equipment

1. Centrifuge 5810R (Eppendorf, catalog number: 022628187)

2. Inverted confocal microscope (Zeiss, model: LSM 800)

Software and datasets

1. Zen 2.3 SP1 FP3 (black) 64-bit (Carl Zeiss), licensed

2. GraphPad Prism Version 10.1.2 (324), licensed

3. ImageJ 1.53t (NIH, https://imagej.net/ij/download.html)

4. Microsoft Excel 2016 (16.0.5422.1000) MSO 32-bit, licensed

5. Adobe Illustrator 28.1 64-bit, licensed

Procedure