Ki67 clone b56

Samples were washed twice in PBS prior to staining with LIVE/DEAD (Life Technologies) for 10 min at room temperature. Samples were then blocked with 5 μg/mL anti CD16/32 (clone 2.4G2; produced in house) and heat‐inactivated mouse serum (1:10) in FACs Buffer (dPBS containing 0.5% BSA (Sigma), 2mM EDTA (Sigma)) cell surface markers for several different cell populations were detected by addition of various flourochrome‐conjugated antibodies (See Supporting Information Table I for list of antibodies used) for 20–30 min on ice. Samples were washed in FACs buffer prior to intracellular staining which was performed following fixation in either 2% formaldehyde for 10 min at room temperature or 1× Fixation/Permeabilization buffer (eBioscience) overnight followed by permeabilization using 1× permeabilization buffer (eBioscience). Antibodies to detect intracellular proteins (including Ki67 clone B56 from BD) were incubated with samples on ice in permeabilization buffer (eBioscience) for 30 min. Samples were acquired using a BD LSR II. Macrophages were gated as CD19⁻Ly6G⁻SigF⁻TCRβ⁻F4/80^hiMHC‐II^lo, Eosinophils were gated as SigF⁺SSC^hiMHC‐II⁻, ILC were gated as CD3⁻CD11c⁻B220⁻Ly6G⁻Ly6C⁻F4/80⁻CD11c⁻NK1.1⁻Ter119⁻CD5⁻CD11b⁻CD49b⁻CD45.2⁺CD90.2⁺.

Nine parameter flow cytometric analysis was performed on 200uL of whole blood. Lymphocytes were assessed by forward and side scatter, and stained for the following: anti-CD19-PECy5 (clone HIB19),anti- CD20- APC-H7 (clone L27), anti-CD10- APC (clone HI10a), anti-CD21- PE (clone B-ly4), anti- CD27- PECy7 (clone M-T271) (all from BD Biosciences, San Jose, CA) and anti-CD38- Alexa Fluor 700 (clone HIT2, Biolegend, San Diego, CA). At least 20,000 CD19+ B cells events were acquired on an LSRII cytometer driven by FACSDiVa version 6.2 software. Analysis was performed using FlowJo Version 10 software. BD TruCOUNT (BD Biosciences) beads were used for absolute counts of CD19+ B cells. For CD86 FITC (clone 2331, BD Biosciences, San Jose, CA) whole blood was stained for 10 minutes in the dark at room temperature, then lysed for 2 minutes with 2mL of BD Lyse (BD Pharmigen, San Diego, CA) and washed with 2mL of 0.1% BSA+ PBS, then fixed with 200uL of BD Stabilizing Fixative (BD Biosciences). For Ki-67(clone B56, BD Biosciences),whole blood was stained as above, but permeabilized with BD Cytofix/Permeabilization (BD Biosciences) on ice for 20 minutes, washed with 2mL of Perm Wash (BD Biosciences) and stained with 20uL of anti-Ki-67 FITC for 30 minutes on ice, washed twice with 2 mL of Perm Wash and fixed.

T47D and MDA-MB-231 (5 × 10⁴) cells were labeled with CellTracker Red CMTP (Molecular Probes) according to the manufacturer's instructions and plated in 8 well culture slides (Corning) in DMEM supplemented with 10% fetal calf serum (FCS) the previous day. Two hours before adding CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, Molecular Probes) labeled human monocytes at a ratio of 1:1 (initial amount of cancer cells:monocytes), the medium was replaced with RPMI without serum. The cells were cultured for two days, fixed with ice-cold methanol, stained with Hoechst and imaged for cell number changes. Five 10× microscopic fields for each donor (n = 3, in triplicates) were counted based on the Hoechst stain, using ImageJ software. Co-localization of green signal with Hoechst stain accounted for monocytes, which were therefore excluded from the analysis.
For Ki67 staining the cells in direct co-culture were left unlabeled and cultured as above. The cells were fixed with methanol and stained for Ki67 (clone B56 1:100, BD Biosciences) according to the above-mentioned protocol. To exclude that the added monocytes did not contribute to the Ki67 signal, the monocytes were cultured in parallel with cancer cell conditioned medium and stained for Ki67.

Flow cytometric analysis was carried out using the following reagents: Live/dead UV Zombie, KLRG1 APC (2F1), CD4 Brilliant Violet 711 (OKT-4), CD28 Brilliant Violet 510 (CD28.2) and CD45RA PECy7 (HI100) all from Biolegend. CD28 Brilliant Violet 786 (U28), CD3 PECF594 (UCHT1), CD8 APC-H7 (SK1), CD57 Alexa Fluor 421 (NK-1), and human cutaneous lymphocyte antigen (CLA) FITC (HECA-452) from BD Biosciences. Cell suspensions were incubated with antibody solutions for 30 min at 4°C for extracellular staining. Intracellular staining for Ki67 (clone B56, BD Bioscience) was performed with Foxp3 Staining Buffer Set (Miltenyi Biotec, Bisley, UK). Cytokine and anti-hTERT (rabbit IgG—Abcam) staining were performed using Fix and Perm Cell Permeabilization Kit (Invitrogen, Paisley, UK) according to the manufacturer's protocol. Samples were processed at Fortessa X-20 cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar). Isotype control staining and fluorescence-minus-one controls were used to set the quadrants.