Igg2a isotype control

Lineage depletion was performed using the kit from Miltenyi Biotech, San Diego, CA according to the manufacturer’s instructions. For transplantation experiments, a single cell suspension obtained from BM of adult or E13.5 fetal liver (FL) donor mice was depleted of red blood cells using the PharmLyse® buffer from BD Biosciences, San Jose, CA. Viable cells were then counted and resuspended at the desired concentration in PBS supplemented with 1% BSA, and transplanted into recipient mice by tail vein injections. For transplantation experiments, 2×10⁶ whole BM or FL cells were injected into lethally irradiated or non-conditioned recipients. For competitive transplants, 1×10⁵ BM cells isolated from heterozygous CD45/1.CD45.2 mice were used as supportive cells.
Homing was performed according to our previously published protocol.[30 (link)]
For transplantation experiments using an anti-IL-11 antibody, lethally irradiated recipients were treated 6 h and 24 h after the first dose of irradiation with IL-11 blocking antibody or IgG2A isotype control (both from R&D Systems, Minneapolis, MN) at the dose of 1 mg/kg of body weight (by tail vein injection).[31 (link)] For these experiments, 3×10⁶ whole BM cells were injected into lethally irradiated recipients by tail vein injection.

Rat anti-mouse IL-17A (Clone #50104, catalog #MAB421) or IgG_2A isotype control (Clone #54447, catalog #MAB006) antibody (R&D Systems, Inc., Minneapolis, MN) was administered weekly via intra-peritoneal injections (200μg/mouse, resuspended in 500μl sterile phosphate buffered saline [PBS]) starting 2 weeks post-HCT.^{19 (link)–21 (link)} Based on pilot studies (data not shown), earlier and higher doses of anti-IL-17A were avoided due to concerns over potential impairment in hematopoietic cell engraftment.

GBM1 cells were cultured at 5x10⁴ cells/well. After 24 hrs, thawed CM and RCM were incubated for 1 hr at 4 °C with 0.75 % anti-human-IFNα, -IFNβ, or -IFNγ blocking antibody (alone or in combination; all Pestka Biomedical Laboratories, PBL) or isotype control (rabbit serum (IFNγ) or sheep serum (IFNα and IFNβ) alone or in combination; both Sigma). Simultaneously, when GBM1 cells were to be treated with anti-human-IFNα/IFNβ blocking antibodies, 1.25 % anti-human IFNα/β receptor chain 2 antibody (PBL), or IgG2a isotype control (R&D Systems) were added to cells for 1 hr at 37 °C. Prepared CM/RCM (including block/isotype) was then added for 4 hrs at 37 °C before medium was removed and replaced with full growth medium. After 24 hrs, cells were harvested and stained for PD-L1 expression as described above.