Fc block clone 2.4g2

B16-F10 cells treated with Navtemadlin or DMSO control, were harvested and single-cell suspensions were incubated with Fc Block (clone 2.4G2, BD Biosciences) for 10 minutes at room temperature to reduce unspecific antibody binding. Intracellular staining to detect p53, p21, Bax, PUMA, BCL-2, and MCL-1 Ps159 was performed using the FoxP3 staining kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, cells were fixed in fixation/permeabilization buffer for 30 minutes at 4°C and washed with permeabilization buffer. Fixed cells were then incubated for 30 minutes at 4°C with antibodies against intracellular proteins: p53-Alexa 647, 1:75 (1C12, Cell Signaling Technology); p21-Alexa 488, 1:100 (F-5);Bax-Alexa 595, 1:100 (B-9); PUMA-PE, 1:100 (B-6) from Santa Cruz Biotechnology; BCL-2-PE-Vio770, 1:10 (REA356, Miltenyi Biotec) and MCL-1 Ps159-PE, 1:50 (REA924, Miltenyi Biotec).
After washing, single-cell suspensions were resuspended in flow buffer (0.5% BSA, 2 μmol/L EDTA in PBS) containing the DNA stain FxCycle Violet (1:2,000 dilution, Thermo Fisher Scientific). A minimum of 30,000 cells were acquired on an ImageStreamX Mk II Imaging Flow Cytometer (Amnis corporation) equipped with 405, 488, 561, 642, and 785 nm lasers at 60 × magnification. Data analyses were performed using IDEAS Software (Amnis Corporation).

Spleens were aseptically isolated from ten mice from each group 4 weeks after challenge. Splenocyte suspensions were prepared in RPMI-1640 culture media supplemented with 10% FBS, after RBC lysis with red blood cell lysing buffer hybrid-max (Sigma-Aldrich, St. Louis, MO, USA), for flow cytometry analysis, antibody secreting cell (ASC) assays and cytokine analysis. Cells were stained with trypan blue (Welgene, Daegu, South Korea) and counted with a hemocytometer chamber under a microscope. Splenocytes from each animal were resuspended in staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1 M PBS). Cells from individual mouse were separately incubated with Fc Block (clone 2.4G2; BD Biosciences, CA, USA) to block non-specific binding at 4°C for 15 min, and then stained at 4°C for 30 min with different combinations of FITC, PE, PE-Cy5, PE-Cy7 or APC conjugated anti-CD3e (145-2c11), anti-CD4 (GK1.5), anti-CD8a (53–6.7) (BD Biosciences, CA, USA). For memory T cell responses, anti-CD44 (IM7) and anti-CD62L (MEL-14) were used (BD Biosciences, CA, USA) as indicated [28 ]. For memory B cell responses, anti-CD45R/B220 (RA3-6B2), anti-CD27 (LG-3A10) and anti-IgG1 (A85-1) were used (BD Biosciences, CA, USA) [29 (link)]. Events were acquired on BD Accuri C6 Flow Cytometer (BD Biosciences, CA, USA) and data were analyzed using C6 Analysis software (BD Biosciences, CA, USA).

Pulmonary leukocytes were obtained as previously described by Oliveira-Brito et al. [69 (link)]. Prior to phenotyping the cell populations in the lungs, the concentrations of the cell suspensions were determined using a Neubauer chamber. Each sample was adjusted to a concentration of 1 × 10⁶ cells/mL, and the cells were thereafter stained with fluorophore-conjugated antibodies. The following antibodies were obtained from BD Pharmingen (San Diego, CA, USA): anti-CD3 (PE-Cy5 rat anti-mouse CD3, clone 17A2), anti-CD11c (Alexa Fluor 488 rat anti-mouse CD11c, clone M1/70), anti-Ly6G (PE rat anti-mouse Ly-6G, clone 1A8), and anti-CD11b (PE rat anti-mouse CD11b, clone M1/70). After incubation for 30 min at 4 °C with 0.5 μg of anti-CD16/CD32 mAb (Fc block, clone 2.4G2, BD Pharmingen), 2.5 μg/mL of the aforementioned antibodies was added and the suspension incubated for 45 min at 4 °C. The cells were washed twice with PBS and fixed with 1% PBS-formaldehyde for further analysis using flow cytometry (Guava EasyCyte™ Mini System).

J558 tumors were resected on day 11 post implantation. Single-cell suspensions were prepared by dissociation and passing cells through a 70-µm filter. Cells (1 × 10⁶) were plated in 96-well plates, treated with Fc block (clone 2.4G2; BD Biosciences), and stained with fluorochrome-conjugated antibodies against surface or intracellular markers (Table S1). Expression of mCD38 among tumor-infiltrating immune subsets was performed using a fluorochrome-conjugated mouse CD38 antibody that was determined to bind a different epitope than the anti-mCD38 treatment antibody. For intracellular staining, samples were fixed, permeabilized, and stained with antibodies (FoxP3 staining buffer set; eBioscience, San Diego, CA, USA). Antibody fluorescence was detected by flow cytometry on Fortessa (BD Biosciences), and the results were analyzed using FlowJo^TM software (FlowJo, Ashland, OR, USA).

Cells were incubated with fixable viability dye (eFluor 455UV; eBioscience, UK) followed by CD16/CD32 antibody (Fc-block; clone 2.4G2, BD Bioscience, UK), then stained with directly conjugated antibodies (10 μg/ml and analysed using an LSR II device (BD Bioscience, UK). The following antibodies were purchased from BD Bioscience, UK unless stated otherwise: MHC class II #553623, CD4 #552051, CD11b #550993, CD11c #550261, CD135 #562537, CD40 #562846, CD86 #560581, CD25 #553866, CCR7 #563596, PDL-1 #564716, Zbtb46 #565832, SIRP-1α #740159, c-kit #560185, CD115 #25–1152-82 (eBioscience) and CX3CR1 #149023 (Biolegend). Transcription factor buffer (BD Bioscience, USA, catalog #562574) was used in accordance with the manufacturer’s instructions to test for transcription factors. FlowJo software (TreeStar, Inc., Ashland, OR, USA) was used for data analysis.