Anti cd45 apc clone 30 f11

Split Neo-2/15 fusion proteins (or intact Neo2 constructs) were administered daily to nontumor-bearing Foxp3-GFP mice (JAX Stock, no. 006772) for 5 days. Neo2A fusions were injected subcutaneously in the scruff of the neck while Neo2B fusions, PBS and intact Neo-2/15 constructs were injected intraperitoneally. Intact Neo-2/15 fusions were dosed at 500 pmol (12 µg per mouse per day). Split Neo-2/15 fusions were dosed at 500 pmol (10 µg per mouse per day) and 250 pmol (4 µg per mouse per day). On day 6 mice were euthanized, spleen and both inguinal lymph nodes collected and homogenized into single-cell suspensions (and spleen resuspended briefly in ACK lysis buffer to lyse red blood cells) and cell populations investigated by flow cytometry using the following antibodies: APC anti-CD45 (clone 30-F11, Biolegend, no. 103111); Brilliant Violet 421 anti-CD4 (GK1.5, Biolegend, no. 100443); and PE anti-CD8a (53-6.7, Biolegend, no. 100707). All antibodies were used at 1:100 dilution.

Splenocytes were harvested from a WT C57BL/6 J mouse and plated together with split Neo-2/15 fusion test items at the indicated concentrations (negative-control, single-split construct wells were plated at 50 nM) in a 96-well plate in RPMI complete medium. After 4 days, cells were harvested and stained for flow cytometry. The antibodies used for staining were: APC anti-CD45 (clone 30-F11, Biolegend, no. 103111); Zombie NIRTM Fixable Viability Kit (Biolegend, no. 423105); Pacific Blue anti-B220 (RA3-6B2, Biolegend, no. 103227); and Brilliant Violet 421 anti-CD4 (GK1.5, Biolegend, no. 100443). All antibodies were used at 1:100 dilution.