A Versatile In Vitro Quantitative Assay for Macrophage Efferocytosis in Diverse Research Applications

Materials and reagents

Reagents

1. Isoflurane (RWD, catalog number: R510-22-10)

2. Annexin V-FITC/PI Apoptosis Detection kit (Elabscience, catalog number: E-CK-A211)

3. CellTrace^TM CMTPX Red cell tracer (Yeasen Biotechnology, catalog number: 40717ES)

4. CFDA SE Cell Proliferation and Cell Tracking kit (Yeasen Biotechnology, catalog number: 40714ES), comprising CFDA-SE fluorescent probe (40714-A), CFDA-SE solvent (40714-B), and 5× cell staining buffer (40714-C)

5. Collagenase Type IV (Sigma, catalog number: C5138, specific activity ≥125 CDU/mg)

6. Dexamethasone (GlpBio, catalog number: GC40775)

7. EDTA (Invitrogen, catalog number: 91222920)

8. M199 medium (GlpBio, catalog number: C11150500BT)

9. Oxidized LDL (ox-LDL) (Yiyuan Biotech, catalog number: YB-002)

10. Penicillin-streptomycin solution (pen/strep) (Procell Life Science & Technology, catalog number: PB180120)

11. RPMI 1640 culture medium (Gibco, catalog number: C11875500BT)

12. Sodium arsenite (Sigma-Aldrich, catalog number: S9663)

13. Thioglycollate medium (Sigma-Aldrich, catalog number: T9032)

14. Phosphate buffer saline (PBS), pH 7.4 (Biopico Life Sciences, catalog number: 090501)

15. 1 M HEPES solution (Procell Life Science & Technology, catalog number: PB180325)

16. 10× Hanks’ balanced salt solution (HBSS), Ca²⁺/Mg²⁺-free (Sigma-Aldrich, catalog number: H4641)

17. Calcium chloride, anhydrous (CaCl₂) (Sangon Biotech, catalog number: A501330)

18. Red blood cell lysis buffer (Elabscience, catalog number: E-CK-A105)

19. Fetal bovine serum (FBS) (Procell Life Science & Technology, catalog number: 164210)

20. Sodium pyruvate solution (Procell Life Science & Technology, catalog number: PB180422)

21. GlutaMAX (Gibco, catalog number: 35050061)

22. Puralube vet ointment (Dechra, catalog number: 1703321138)

Solutions

1. 3% (w/v) thioglycollate medium (see Recipes)

2. 1,000× CFDA-SE stock solution (see Recipes)

3. CFDA-SE working solution (see Recipes)

4. Wash buffer (see Recipes)

5. Collagen buffer (see Recipes)

Recipes

1. 3% (w/v) thioglycollate medium

Prepare fresh 3% (w/v) thioglycollate medium aseptically in a biosafety cabinet by dissolving 0.03 g of thioglycollate medium powder (4 °C storage) in 1 mL of sterile PBS. Immediately apply the prepared solution to maintain optimal induction efficiency.

2. 1,000× CFDA-SE stock solution

To prepare the 1,000× CFDA-SE stock solution, add 500 μL of CFDA-SE solvent to one tube of CFDA-SE fluorescent probe (1.4 mg) and mix thoroughly to yield a clear stock solution with a concentration of 2.8 mg/mL. Aliquot the stock solution immediately after preparation and store it at ≤ -20 °C (or -70 °C for an extended shelf life) under dry and light-protected conditions; repeated freeze-thaw cycles must be strictly avoided. The stock solution is optimal for use within 1 month and has a maximum shelf life of 2 months when stored as specified above.

3. CFDA-SE working solution

Prepare 1× cell staining buffer (the diluent for CFDA-SE) by taking the supplied 5× cell staining buffer and diluting it 1:5 with sterile deionized water (e.g., 2 mL of 5× buffer mixed with 8 mL of sterile deionized water) with thorough agitation. Store the diluted 1× buffer at 4 °C, protected from light, and use it within 1 week for optimal performance. For the 2× CFDA-SE working solution, take the 1,000× CFDA-SE stock solution thawed to 20–25 °C, dilute it 1:500 with freshly prepared 1× cell staining buffer (e.g., 2 μL of 1,000× stock added to 1 mL of 1× buffer), and mix gently to form a homogeneous 2× working solution.

Critical: The working solution must be freshly prepared immediately before use, as CFDA-SE is susceptible to hydrolysis in aqueous solutions; all preparation steps should be performed in the dark to prevent fluorophore quenching. All kit components are pre-sterilized, and no additional sterilization is required.

4. Wash buffer

Prepare the solution according to the table above and autoclave before use.

5. Collagen buffer

Prepare the solution according to the table above and autoclave prior to use.

Note: The 50 mM CaCl₂ working solution is prepared by diluting 2 mL of 1 M CaCl₂ stock solution (11.1 g of CaCl₂ dissolved in 100 mL of ddH₂O) into 38 mL of ddH₂O. Due to the poor aqueous stability of collagenase, add 30 mg of collagenase powder to 30 mL of the pre-autoclaved solution immediately before primary hepatocyte isolation, yielding a working concentration of 1 mg/mL (≥125 CDU/mL) to ensure on-site preparation and immediate application.

Laboratory supplies

1. Scissors (Beyotime, catalog number: FS500)

2. Tweezers (Beyotime, catalog number: FS500)

3. 1 and 5 mL syringes (Heshou, Changzhou Yuekang Medical Devices Co., Ltd.)

4. 70 μm cell strainer (BIOFIL, catalog number: 01.TC.010.0053)

5. 15 mL centrifuge tube (BIOFIL, catalog number: CFT011150)

6. P1250 pipette tips (A-gen Biotechnology, catalog number: T-1250-B)

7. P200 pipette tips (Biosharp, catalog number: BS-200-T)

8. P10 pipette tips (Axygen, catalog number: T-300)

9. 24 G catheter (Yikang group)

10. 35 mm non-treated glass-bottom cell culture dish for laser confocal microscopy (BIOFIL, catalog number: BDD012035)

11. 60 mm cell culture dish (BIOFIL, catalog number: TCD000060)

Equipment

1. Automatic cell counter (Thermo Fisher Scientific, model: Countess 3)

2. Confocal laser scanning microscope (Nikon, model: A1R)

3. Flow cytometer (BD, model: FACSCelesta)

Software and datasets

1. FlowJo software (Version X; TreeStar, Ashland, OR, USA)

2. GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA)

Procedure