70 m nylon cell strainer

Splenic B cells were isolated from C57BL/6NRj (wt) mice or b12 [29 (link)] and PGT121 [28 (link)] B cell receptor-transgenic mice by magnetic cell separation (MACS; B Cell Isolation Kit (mouse), 130-090-862, Miltenyi Biotec, Bergisch-Gladbach, Germany) following the manufacturer’s guidelines. For preparation, the spleens were homogenized with a gentleMACS dissociator (Miltenyi Biotec, Bergisch-Gladbach, Germany) and filtered through a 70 µm nylon cell strainer (Fisher Scientific, Hampton, NH, USA). The cells were then incubated for 10 min at RT in ACK buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) for erythrocyte lysis. The splenocytes were washed once in R10 medium before MACS separation was performed.

C57BL/6 mice of 6–8 weeks of age were euthanized by cervical dislocation. Animal experiments were performed in accordance with standard ethical guidelines and approved by the regional laboratory animal ethics committee in Malmö/Lund (Permit No. M36-13). Femoral and tibial bones were dissected. Skin and excess muscle tissue were removed. Femur and tibia were separated by a cut at the knee joint and bone marrow was flushed with 10 ml of phosphate-buffered saline (PBS; Life Technologies, Carlsbad, CA, USA) supplemented with 2% heat-inactivated fetal bovine serum (HI-FBS; Life Technologies, Carlsbad, CA, USA). Bone marrow was passed through 70 µm nylon cell strainer (Fisher Scientific, Lund, Sweden), centrifuged and resuspended and incubated in ammonium-sodium-chloride lysis buffer (ACK; Life Technologies, Carlsbad, CA, USA) for 5 min to lyse red blood cells. Cells were then washed with PBS with 2% HI-FBS, centrifuged and resuspended in RPMI 1640 culture medium supplemented with 10% HI-FBS and 1% penicillin-streptomycin.
For additional experiments, the human monocytic leukemia cell line THP-1 (InvivoGen, San Diego, CA, USA) was cultured in RPMI 1640 culture medium supplemented with 10% HI-FBS and 1% penicillin-streptomycin.

For murine orthotopic tumor studies, we used samples collected from a previously published study [34 (link)]. Briefly, 10⁶ syngeneic luciferase-expressing KPC tumor cells, originally derived from a primary pancreatic tumor in a KPC mouse (PDX-1-Cre, LSL-Kras^G12D, LSL-Trp53^−/−), were injected in Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) into the tail of the pancreas of C57BL6/J mice. Bioluminescent imaging was used to monitor tumor growth, and tumor weight was measured at time of sacrifice. Three (3) weeks following orthotopic injections when mice had terminal tumor volume by imaging and weights, mice were euthanized, and spleens from tumor-free and tumor-bearing animals were removed and placed in PBS on ice. Under sterile conditions, the spleens were mashed with a 1 mL syringe plunger and strained twice through a 70 µm nylon cell strainer (Fisher Scientific, Waltham, MA, USA). The cells were centrifuged at 1700 rpm for 5 min before aspirating the supernatant. Splenocytes were resuspended in 10 mL red blood cell lysis buffer and pipetted to ensure total lysis of the red blood cells. The cells were resuspended in a freezing medium (90% FBS, 10% DMSO) and stored in liquid N₂ prior to flow cytometry staining. All splenocytes collected from the prior study [34 (link)] were cryopreserved until use in this study.