Mab453 500

The 24-well plate and 3 μm polyester membrane inserts were coated with 2% BSA solution for 1 hour at 37°C, followed by washing with DPBS (GIBCO). 0.4×10⁶ differentiated PLB-985 cells or mouse primary neutrophils (in 100 μl HBSS) were seeded in the upper chamber. 600μl conditioned media was added into the lower chamber. For CXCL1 blockage, 1 μg/ml anti-CXCL1 antibody (MAB453-500, R&D Systems) was added into the lower chamber. After incubating at 37°C for 2 hours, the membrane was stained and cell numbers in the lower chamber were counted.

To generate the genetically engineered pancreas specific Kdm6a knockout (KO) mouse model, we first crossed Ptf1a^Cre mice (kindly shared by Dr. Christopher Wright) with Kdm6a^fl/fl (female) or Kdm6a^fl/Y (male) mice (Jackson Laboratory, #024177) to get Ptf1a^Cre Kdm6a^fl/fl (female homozygous Kdm6a KO) or Ptf1a^Cre Kdm6a^fl/Y (male hemizygous Kdm6a KO). Then we crossed LSL-Kras^G12D/wt;LSL-p53^R172H/wt (kindly shared by Dr. Tyler Jacks) with Ptf1a^Cre Kdm6a^fl/fl or Ptf1a^Cre Kdm6a^fl/Y to generate five different genotypes of mice (KPC- Kdm6a^Y/+, KPC- Kdm6a^Y/fl, KPC- Kdm6a^+/+, KPC- Kdm6a^+/fl and KPC- Kdm6a^fl/fl). All the animal studies were performed under the reviewed and approved animal protocol (#PRO00007450) by the Unit of Laboratory Animal Medicine (ULAM) at the University of Michigan.
For orthotopic mouse models, 5×10⁴ KPC 7940 cells (a gift from Dr. Gregory Beatty) in 40 μl Matrigel and cell culture media (1:1) were injected into the pancreata of 6- to 8-week-old C57BL/6J mice. IgG or anti-CXCL1 antibody (4 mg/kg, MAB453-500, R&D Systems) was intraperitoneally injected twice a week from the 8th day of tumor injection. Four weeks after the tumor cell injection, mice were euthanized, and tumor tissues were collected and fixed in formalin.