Heat inactivated fetal calf serum

Dulbecco’s minimum essential medium Eagle (DMEM), penicillin, streptomycin and fungizone were purchased from Highveld Biological (Pty) Ltd. (Sandringham, SA). Sterile cell culture flasks and plates were obtained through Lasec SA (Pty) Ltd. (Honeydew, Johannesburg, South Africa). Gibco® collagenase and hyaluronidase were purchased from Life Technologies (Johannesburg, Gauteng, South Africa). A lactate dehydrogenase cytotoxicity Assay Kit II kit from BioVision Inc. (Mountain View, California, USA) was supplied by BIOCOM biotech (Pty) Ltd. (Clubview, South Africa). All other chemicals of analytical grade, as well as heat-inactivated fetal calf serum (FCS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Ported PCL particles were manufactured according to a previously published protocol (18). Non-ported PCL particles (i.e. PCL particles with no ports) were manufactured as per the published protocol with the omission of the port forming reagents. In a previously unpublished study in rats, we observed that the biotoxicity of PCL particles in vivo is low to non-existent, and where present is reflective of a foreign body response (Figs A-E in S1 File and Table A in S2 File, all raw data is in S3 File). PS particles (mean particle diameter of 150 μm) were obtained from Corpuscular Inc. (Cold Spring, New York, USA).

Bovine serum albumin, thiobarbituric acid (TBA), phenazine methosulphate (PMS), nitroblue tetrazolium (NBT), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), 3-(4,5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2–7-diacetyl dichlorofluorescein (DCFH-DH), rhodamine 123 (Rh-123), acridine orange, ethidium bromide, heat inactivated fetal calf serum (FCS), minimum essential medium (MEM), McCoy’s modified medium, glutamine, penicillin-streptomycin, EDTA and trypsin were purchased from Sigma Chemicals Co. (USA).

HT22 cells (kindly provided by David Schubert, Cellular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA) were grown in Dulbecco’s modified Eagle medium (DMEM, Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 10% heat-inactivated fetal calf serum (Merck KGaA, Darmstadt, Germany), 100 U/mL penicillin, 100 mg/mL streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 2 mM L-glutamine (Merck KGaA, Darmstadt, Germany). To induce cell death, erastin (Calbiochem^®, Merck KGaA, Darmstadt, Germany) was added to the medium for the indicated amount of time (8–16 h).

Isolated splenic CD4⁺ T cells were cultivated in an RPMI 1640 medium that was supplemented with 10% heat-inactivated fetal calf serum (Merck, Darmstadt, Germany), antibiotics (penicillin and streptomycin mixture, Fujifilm Wako Pure Chemicals), Con-A (5 µg/mL), and IL-2 (10 U/mL) for 48 h. Membrane potentials were measured by using the voltage-sensitive dye DiBAC₄(3), as previously reported [9 (link),38 (link)]. Changes induced in the fluorescent intensity of DiBAC₄(3) by alkaline pH (pH 8.5) were measured with an ORCA-Flash2.8 digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Data collection and analyses were performed by using an HCImage system (Hamamatsu Photonics). Images were obtained every 5 s.

To determine activation of primary NK cells following exposure to target cells pulsed with different peptide variants, NK cell degranulation assays measuring CD107a expression were performed using freshly isolated and purified NK cells from nine healthy KIR2DL3+ individuals [38 (link)]. Primary NK cells were isolated by incubating whole blood with RosetteSep Human NK Cell Enrichment Cocktail (Stemcell Technologies) for 20 min at room temperature, followed by Histopaque-1077 (Sigma) density gradient centrifugation. NK cells were rested overnight in RPMI medium 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (Sigma-Aldrich), 2,500 U/ml penicillin, 2,500 μg/ml streptomycin, 100 mM L-glutamine (Cellgro), and 1.0 ng/ml IL-15 (Cellgro). Subsequently, NK cells (1 × 10⁵) were co-incubated with peptide-pulsed 721.221-ICP47-C*03:04 cells (5 × 10⁵) at an effector:target ratio of 1:5 in RPMI containing anti-human CD107a-PE-Cy7 (12.5 μl/ml) and monensin (1.5 μl/ml, BD GolgiStop). Cells were incubated for 6 h at 26°C in 5% CO₂. Cells were stained with 7AAD, anti-CD3-PB, anti-CD16-BV785, anti-CD56-BV605, anti-CD14/19-BV510, anti-KIR2DL2/3-PE, and anti-KIR2DL3-APC, washed, fixed with 4% (w/v in PBS) paraformaldehyde (Affymetrix), and analyzed by multiparameter flow cytometry using a BD LSR II.

The THP1 acute monocytic leukemia cell line (DSMZ ACC 16) was selected as a model for monoblasts and macrophages (after in vitro differentiation, dTHP1), whereas the HT-29 human colon adenocarcinoma cell line (DSMZ ACC 299) was chosen as a non-hematopoietic lineage tumor model. THP1 and HT-29 cells were cultured in RPMI 1640 (Lonza, Basel, Switzerland) and McCoy's medium (Sigma-Aldrich, St. Louis, MO/US), respectively. Both media were supplemented with 10% heat-inactivated fetal calf serum (Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin and 1% GlutaMAX™ Supplement (Gibco, Gaithersburg, MD/US). To induce differentiation into macrophages, THP1 cells were treated with 200 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 72 h and rested in a PMA-free medium for 5 days as described by Daigneault et al. (25 (link)). All cell cultures were maintained at 37°C in a humidified atmosphere and 5% CO₂.