Materials and Reagents

1. Mice
   Any donor mice can be used, as long as the donor and recipients have the same background strain (i.e. BALB/C into BALB/c or Bl/6 into Bl/6) to prevent rejection issues. We selected transgenic donor mice that had an increased frequency of B cells specific for our antigen of interest, NP. This way we could be certain of the number of B cells specific for our antigen and these would be easy to identify by flow cytometry and elispot. However, wild-type mice will also respond to NP, just at a lower frequency.
   
   1. B1.8^+/-Jκ ^+/- BALB/c mice
      Note: B1.8 KI BALB/c mice were generated as described (Sonoda et al., 1997) and maintained on the Jκ KO strain (Chen et al., 1993) to enrich the frequency of λ⁺ NP-specific B cells. B1-8 KI ^+/+ Jκ KO ^-/- mice were crossed to BALB/c mice from The Jackson Laboratory (Bar Harbor, ME) to generate B1.8^+/-Jκ ^+/- BALB/c mice, which were used for naïve controls and for transfers of NP⁺ B cells used to generate MBCs.
      
   2. AM14 Tg x Vκ8R KI BALB/c mice were generated as described (Shlomchik et al., 1993; Hannum et al., 1996; Prak and Weigert, 1995), which were used as recipient mice for primary immunization
      Note: All mice were maintained under specific pathogen-free conditions. The Yale Institutional Animal Care and Use Committee approved all animal experiments.
   
   
2. Immunizations
   For generating memory B cells in a primary response, mice were immunized intra-peritoneally with 50 µg of 4-hydroxy-3-nitrophenyl acetyl (NP)-Chicken Gamma Globulin (CGG) precipitated in alum. The ratio of NP to CGG ranged between 26 and 33. All mice were immunized at 6-12 week of age
   
   
3. Isolation of B cells from donor mice
   
   1. 2 pairs sterile scissor and forceps
      
   2. Sterile frosted slides
      
   3. Sterile Petri dishes
      
   4. Autoclaved Pasteur pipettes
      
   5. 70% ethanol
      
   6. Sterile ACK (RBC lysing buffer) (Lonza, catalog number: 10-548E )
      
   7. 100 µM filter (BD Biosciences, catalog number: 340615 )
      
   8. Ice
      
   9. Conical tubes (14 ml v-bottom) (BD Biosciences, Falcon^®)
      
   10. Falcon 14 ml polystyrene round-bottom tubes (BD Biosciences, catalog number: 352057 )
       
   11. Trypan blue solution (0.4%) (Life Technologies, catalog number: 15250-061 )
       
   12. EasySep™ Mouse B Cell Enrichment Kit (STEMCELL Technologies, catalog number: 19754 ). Components of kit:
       
       1. EasySep™ (Negative Selection) Mouse B Enrichment Cocktail, 0.5 ml
          
       2. EasySep™ Biotin Selection Cocktail, 1 ml (store at 4 °C)
          
       3. EasySep™ Magnetic Particles, 1 ml (store at 4 °C; turn centrifuge on and cool to 4 °C)
          
       4. Normal Rat serum, 1 ml (store at -20 °C)
   13. RPMI-1640 with L-glutamine (Sigma-Aldrich, catalog number: R8758 )
       
   14. Fetal Calf Serum (GE Healthcare HyClone^TM)
       
   15. HEPES 1 M (Corning Incorporated, catalog number: 25-060-Cl )
       
   16. Streptomycin/penicillin, 10,000 U/ml (Life Technologies, Gibco^®, Catalog number 15140-122 )
       
   17. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3128 )
       
   18. PBS without Ca²⁺/Mg²⁺ (Life Technologies, Gibco^®, catalog number: 10010-023 )
       
   19. Ethylenediaminetetraacetic Acid (EDTA) 0.5 M Solution (pH 8) (Thermo Fisher Scientific, catalog number: 25783 )
       
   20. 2.5% Anticoagulant citrate dextrose solution [ACD(A)] (Polymed, catalog number: 7300 )
       ACDA was from the blood bank (http://www.polymedicure.com/?wpcproduct=acd-bag). Each 100 ml of ACD solution-A contains 2.2 g sodium citrate, 0.73 g citric acid, 2.45 g dextrose and 100 ml water.
       
   21. NP-binding reagents: NP-allophycocyanin (APC) (Shlomchik lab)
       
   22. Anti-CD4 (GK1.5) (Shlomchik lab)
       
   23. anti-Fc gamma RIII/II (2.4G2) (Shlomchik lab)
       
   24. anti-CD19 (1D3.2) (Shlomchik lab)
       
   25. 27 G needle, 1 ml syringe
       
   26. Ethidium Monoazide (EMA) 2 mg/ml (Molecular Probes)
       
   27. Complete media (see Recipes)
       
   28. EasySep media (see Recipes)
       
   29. Transfer buffer (see Recipes)
       
   30. Staining Media (see Recipes)

Equipment

1. Sterile hood
   
2. Refrigerated table top centrifuge
   
3. Hemocytometer
   
4. “EasySep” magnet (max vol 8 ml; min vol 250 µl) (STEMCELL Technologies, catalog number: 18001 )
   

Procedure