Evaluation of B Cell Proliferation in vivo by EdU Incorporation Assay

Materials and Reagents

1. BD Micro-Fine^TM Plus 0.5 ml 30 G insulin syringe (BD, catalog number: 230-45094 )
2. Sterile Syringe 3 ml, luer lock (MedHarmony, catalog number: 181110 )
3. Monoject^TM 18 G blunted cannula (Covidien, catalog number: 8881202348 )
4. Cell strainer with a 70 µm pore size (SPL, catalog number: 93070 )
5. 5 ml polystyrene round-bottom tube with a cell strainer cap (Falcon, catalog number: 352235 )
6. 8-week old C57BL/6 wild-type mice (Envigo)
7. 5-ethynyl-2’-deoxyuridine (EdU) (Thermo Fisher Scientific, Invitrogen, catalog number: E10187 )
8. Bovine serum albumin (BSA), fraction V (MP, catalog number: 160069 )
9. Click-iT^TM Plus EdU Alexa Fluor^TM 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific, Invitrogen, catalog number: C10634 )
10. TruStain FcX^TM (anti-mouse CD16/32) Antibody (Biolegend, clone: 93, catalog number: 101319 )
11. V500 anti-mouse B220 (CD45R) antibody (BD, clone: RA3-6B2, catalog number: 561226 )
12. Brilliant violet 605 anti-mouse CD138 (Syndecan-1) antibody (Biolegend, clone: 281-2, catalog number: 142515 )
13. PE-Cy7 anti-mouse CD95 (FAS) antibody (BD, clone: Jo2, catalog number: 557653 )
14. Alexa Fluor 700 anti-mouse CD38 antibody (eBioscience by Thermo Fisher Scientific, clone: 90, catalog number: 56-0381-82 )
15. Biotin anti-mouse IgA antibody (Biolegend, clone: RMA-1, catalog number: 407003 ). Detected with Streptavidin APC-Alexa Flour 780 (eBioscience by Thermo Fisher Scientific, catalog number: 47-4317-82 )
16. Brilliant violet 421 anti-mouse CD196 (CCR6) antibody (BD, clone: 140706, catalog number: 564736 )
17. Phosphate buffered saline (PBS) (Biological Industries, catalog number: 02-023-1A )
18. 1% BSA PBS (see Recipes)
19. EdU solution (see Recipes)
    

Equipment

1. Dry bath incubator
2. Analitical balance
3. Rocking shaker
4. Curved scissors (FST, catalog number: 14091-09 )
5. CytoFlex flow cytometer (Beckman Coulter)
   

Software

1. FlowJo software (LLC)

Procedure

Notes: Buffers should be prepared fresh on the day of the experiment:

1. 1x Click-iT^® EdU permeabilization and wash reagent is diluted with deionized water.
2. Click-iT^® EdU buffer additive aliquots should be kept in -20 °C and diluted only prior to the preparation of the reaction cocktail with deionized water.
3. Mice used in this protocol were 8-10 weeks old with an average weight of ~25 g.
4. All animal experiments should be performed with an adequate ethics approval.
5. This protocol was used to measure B cell proliferation within Peyer’s patches but it is also suited for other lymph nodes and spleen.
   
   

1. Inject 200 µl EdU (2 mg) intravenously via the retro-orbital sinus of C57BL/6 wild-type mice.
2. 2.5 h following EdU administration, sacrifice the mice by cervical dislocation.
3. Open the abdominal cavity and remove the small intestine from the cecum to stomach.
4. Flush the intestine with 40 ml PBS using a 30 ml syringe with an 18 G Monoject^TM blunted cannula. Avoid contact of the tissue with the flushed out intestinal contents.
5. Using curved scissors facing up, excise the Peyer’s patches and avoid excising adjacent intestinal tissue (about 6-10 Peyer’s patches are expected per mouse with an overall of 3-10 million cells from all Peyer’s patches).
6. Place the PPs in a petri dish with a 70 µm cell strainer and 3 ml 1% BSA PBS.
7. Using a pistol of a 3 ml syringe mash the PPs to create a single-cell suspension of PP-derived cells.
8. Transfer the cell suspension into a FACS tube with a cell-strainer cap.
9. Wash the petri dish with additional 1 ml of 1% BSA PBS buffer and collect the solution into the same FACS tube to maximize cell collection.
10. Spin down the cells at 300 RCF for 7 min at 4 °C.
11. Discard the supernatant and resuspend the cells with the residual buffer (approximately 100 µl).
12. Add 1 µl Fc blocker antibody (TruStain FcX^TM, CD16/32), vortex and place on ice.
13. Incubate 5 min on ice.
14. Add 100 µl staining mix prepared in 1% BSA PBS. If proliferation should be analyzed in the B cell compartments, as in the associated study, the following markers can be used: the germinal center (B220⁺ CD138^- FAS⁺ CD38^-); the subepithelial dome (B220⁺ CD138^- IgA⁺ CD38⁺ CCR6⁺) and the plasma cell compartment (CD138⁺). As negative control, we assessed EdU incorporation in the Naïve B cell compartment (B220⁺ CD138^- CD38⁺ FAS^-). Since the residual volume in the tube following blocking is approximately 100 μl, the antibody mix is prepared as a 2x concentration to achieve final dilution as indicated in Table 1.
    
    Table 1. List of antibodies used for surface staining of GC and SED B cells
    
    
    
15. Vortex and incubate on ice for 30 min in the dark to allow surface marker staining.
16. Wash the cells with 2 ml 1% BSA PBS and spin down the cells at 300 RCF for 7 min at 4 °C.
17. Resuspend the cells with 100 µl Click-iT^TM fixative (4% paraformaldehyde in PBS).
18. Incubate for 15 min in the dark at room temperature.
19. Add 3 ml 1% BSA PBS and spin down the cells at 300 RCF for 7 min at 4 °C.
20. Discard the supernatant and resuspend the cells with 100 µl 1x Click-iT^® EdU permeabilization and wash reagent.
21. Mix well by pipetting, do not vortex.
22. Incubate for 15 min in the dark at room temperature.
23. Prepare the Click-iT^TM Plus reaction cocktail according to the Table 2.
    
    Table 2. Click-iT^TM Plus reaction cocktail
    
    Important notes:
    
    1. Prepare the cocktail during Step 22.
    2. Add the reaction components in the order listed in the table.
    3. Use the cocktail within 15 min of its preparation.
    4. Keep the cocktail at room temperature until use.
    5. Extra volume calculation is not necessary.
    
    
24. Add 500 µl of the Click-iT^TM Plus reaction cocktail to each sample. The total volume is 600 µl.
25. Incubate for 30 min in the dark at room temperature.
26. Add 3 ml 1x Click-iT^® EdU permeabilization and wash reagent and spin down the cells at 300 RCF for 7 min at 4 °C.
27. Resuspend the cells with 500 µl 1x Click-iT^® EdU permeabilization and wash reagent and analyze the cells using CytoFlex flow cytometer (Beckman Coulter).
    

Data analysis

Cells that incorporated the EdU are detected by flow cytometry with the indicated fluorophore. Gate on your population of interest and from this population gate on the EdU⁺ cells in a histogram or on a dot plot. EdU⁺ cells in the germinal center are often a distinct population (see Figure 1 below) but in some cases may appear as a tail of the population, depending on the detection efficiency. Naïve B cells are used as a negative control, where no proliferation takes place. For 2.5 h incubation with EdU, the normal range for GC EdU⁺ cell population is 20-30%. A total of 1.5 million events were acquired per sample.


Figure 1. EdU distribution in naïve, GC and SED PP B cells

Notes

This protocol provides details for EdU detection using a commercial kit by Invitrogen, Thermo Fisher Scientific and was therefore adapted from the kit manual and adjusted to our experimental setup. Please note that two flow cytometry kits are available: Click-iT^TM EdU Flow Cytometry Assay Kit and Click-iT^TM Plus EdU Flow Cytometry Assay Kit. This protocol is reproducible only with the Click-iT^TM Plus kit due to its compatibility with the fluorophores used in the study associated with this protocol.

Recipes

1. 1% BSA PBS
   1 g BSA, fraction V
   100 ml PBS
   Dissolve on a rocking shaker. Do not vortex
   Prepare fresh and keep on ice
2. EdU solution (10 mg/ml)
   
   1. Weigh 10 mg EdU in an Eppendorf tube
   2. Dissolve in 1 ml PBS and incubate at 37 °C in a dry bath until EdU is mostly dissolved.
   Note: EdU solution was usually prepared fresh but can be stored at -20 °C for reuse. Reuse of dissolved EdU solution might require reheating at 37 °C to improve its solubility.
   

Acknowledgments

Z.S. is supported by the European Research Council (grant No. 677713), Human Frontiers of Science Program (grant No. CDA-00023/2016), Israel Science Foundation (grant No. 1090/18), Azrieli Foundation, Rising Tide Foundation and the Morris Kahn Institute for Human Immunology. Z.S. is a member in the European Molecular Biology Organization Young Investigator Program and is supported by grants from The Benoziyo Endowment Fund for the Advancement of Science, The Sir Charles Clore Research Prize, Comisaroff Family Trust, Irma & Jacques Ber-Lehmsdorf Foundation, Gerald O. Mann Charitable Foundation and David M. Polen Charitable Trust.
  This protocol provides details regarding EdU in vivo proliferation assay as previously described (Biram et al., 2019).

Competing interests

The author declare no competing interests.

Ethics

All experimental procedures have been approved by the Weizmann Institute Animal Care and Use Committee (IACUC) and followed all relevant ethical regulations, IACUC number 00960118-4.

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