Guava viacount assay

Human fetal astrocytes (passage 1) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in ambient O₂ and 5% CO₂ as previously described (Bitto et al., 2010 (link); Bhat et al., 2012 (link)). In order to induce premature senescence via oxidative stress, cells were seeded at standard density (1 × 10⁴ cells/cm²) and the following day treated with 200 μM hydrogen peroxide (H₂O₂) for 2 h. Cells were considered senescent at least 5 days after the initiation of treatment, as verified previously, (Bitto et al., 2010 (link)) and in subsequent quantitative real-time PCR (qRT-PCR) experiments by increases in senescence marker p21, flattened and enlarged morphology, and cessation of division, and were harvested 7 days after treatment. Viability of senescent astrocytes was not significantly different than the controls (92% ± 1 vs. 95% ± 2.7; p = 0.08) as measured by the Guava ViaCount assay (EMD Millipore).

Cells were seeded in 96-well plates at 2000 cells/well in 100 μL of medium prior to virus infection. The infectious shRNA lentivirus was diluted in medium containing 10 μg/mL polybrene. Medium was removed from the cells, and serial dilutions of 100 μL of lentivirus were added to each well. After 24 hours, virus was removed and replaced with 100 μL of fresh medium. For experiments using the inducible shRNAs, fresh medium containing 20 ng/mL doxycycline was added. Cell viability was measured 5 days after infection using the CellTiter-Blue reagent (Promega, Madison, WI). The CellTiter-Blue reagent was diluted at a ratio of 1:4 in medium, and 20 μL of the diluted reagent was added to each well and incubated at 37°C for 2 hours before recording fluorescence (531_Ex/595_Em) on an Envision 2104 multilabel reader (Perkin Elmer, Waltham, MA). Live cells were counted using a Guava ViaCount assay as described in the manufacturer’s protocol (EMD Millipore, Billerica, MA).

Cell cycle distribution of each culture was determined with propidium iodide flow cytometry, as described previously [21 (link)]. A portion of the cell suspension was used to determine both absolute cell numbers and the fractions of viable and dead cells using the Guava ViaCount assay (EMD Millipore, Etobicoke, Ontario, Canada). Statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, La Jolla, CA,USA).

Blood was obtained by cardiac puncture or submandibular bleed; spleens were excised and homogenized after euthanasia according to approved protocols. Following RBC lysis in ACK buffer, samples were blocked, labeled with antibodies, and analyzed on an LSR II (BD) to assess myeloid cell frequencies. Intracellular Ki67 levels were measured using the Fixation and Permeabilization Buffer Set (eBioscience) and were compared to cells labeled with an isotype control antibody. Antibodies are listed in S3 Table. The Annexin V Apoptosis Detection Kit (eBioscience) was used with propidium iodide to quantify apoptotic cells. For EdU labeling, mice were injected i.p. with 750μg EdU 3 h prior to sacrifice, and the Click-It EdU Kit (Thermo Fisher) was used to detect EdU in splenocytes according to the manufacturer’s protocol.
Myeloid cell definitions were as follows: red pulp macrophages and bone marrow macrophages (Ly6G^- Ly6C^- CD11b^lo F4/80⁺); nonclassical monocytes (Ly6G^- Ly6C^- CD11b⁺ F4/80^int SSC^lo); classical monocytes (Ly6G^- Ly6C⁺⁺ CD11b⁺ F4/80^int SSC^lo) (S1A Fig). Additional labeling with CD115 and CD68 was performed to confirm the identities of monocyte and macrophage populations (S1B and S1C Fig). Absolute cell numbers were quantified in blood prior to RBC lysis using the Guava Viacount assay (EMD Millipore) and in RBC-lysed spleen samples using a hemocytometer.

HMVECs were plated at 2.5 × 10⁴ cells/cm² on p100 tissue culture plates, grown to confluence and subjected to 30 μM of the treatment group’s respective drug, which affects the proliferation of HMVECs due to their angiogenic properties [10 (link)]. The 30-μM treatment concentrations were previously determined to influence the greatest increase in HMVEC proliferation [13 ]. Treatment groups were PNF-1, SC-3-141, SC-3-143, and SC-3-263, with VEGF and endostatin as the positive and negative controls, respectively. Total cells were counted via flow cytometry using the Guava ViaCount Assay (www.millipore.com) immediately prior to treatment and at the 24-h endpoint. Twenty-four hours after stimulation, total RNA was isolated using an RNeasy isolation kit (Qiagen, Inc.) according to the manufacture’s protocol.