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MB Sample ID: SA186791

Local Sample ID:	NecJ 8h 3
Subject ID:	SU002077
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Treatment:

Treatment ID:	TR002089
Treatment Summary:	Apoptotic target cells Live Jurkat cells in RPMI-10 were exposed to 60,000 J of UV energy on a UV Stratalinker 2400 (Stratagene), then incubated at 37C for 4h to generate apoptotic Jurkat cells (ApoJ). Thymocytes, obtained by crushing WT murine thymi and filtering for a single cell suspension, were converted to apoptotic thymocytes in the same manner. For TAMRA-labeling, apoptotic targets were stained during the final hour by 15 minute incubation at 37C with 10M 5(6)-TAMRA,SE (Invitrogen) in PBS followed by two washes with RPMI-10 to remove excess TAMRA. Apoptotic targets were resuspended in fresh RPMI-10 and added to macrophages in culture at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2x106 ApoJ were injected IP. Other cell targets Jurkat cells destined to be IgGJ and NecJ were TAMRA-labeled prior to opsonization or killing using the procedure described above. Labeled cells were incubated for 30 minutes on ice with 25g/mL purified anti-human CD3 (Biolegend, Clone UCHT1) to generate IgGJ or incubated for 25 minutes at 55C to generate NecJ. All targets were resuspended in RPMI-10. IgG targets were added to macrophages at a ratio of 4:1 and co-cultured for 1h, NecJ targets were added to macrophages at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2x106 IgGJ were injected IP. Non-cell targets 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids Inc.) were used to generate PS-containing liposomes. Lipids suspended in chloroform were mixed at 70:30 POPC:POPS, then chloroform was evaporated under nitrogen. Dry lipids were resuspended in RPMI for a final concentration of 500M lipid and sonicated for 15 minutes until liposomes were formed and solution transitioned from opaque to clear. Liposomes were added to macrophages at a final concentration of 50M in RPMI-10. For other experiments, Flash Red 5m carboxylated polystyrene beads (PS/DVB-COOH·(660,690) carboxyl polystyrene beads, Bangs Laboratories Inc.) were added to macrophages at a final ratio of 1:2. Macrophages were co-cultured with non-cell targets for 1h and unengulfed targets were removed by washing macrophages three times with cold 1X PBS. Isotope-labeled metabolite use For some experiments, Jurkat T cells were cultured for 4 days in media containing [13C,15N]cell-free amino acid mixture (Sigma-Aldrich) (RPMI-1640 medium for SILAC, 10% v/v dyalized FBS, 1X Pen/Strep, 1mM HEPES, 4% v/v amino acid mixture). For some experiments, macrophages were cultured for 7 hours in media containing [13C,15N]Arginine (Cambridge Isotope Laboratories): RPMI-1640 medium for SILAC, 10% dyalized FBS v/v, 1X Pen/Strep, 1mM HEPES, 100M Sodium Pyruvate, 0.2 g/L [13C,15N]Arginine. FACS sorting engulfing and non-engulfing cells Single cell suspensions were incubated with unlabeled CD16/CD32 for 5 min to block non-specific FcR-mediated binding. Cells were stained with surface antibody panels for 20 min, and then washed. Cells were protected from light and incubations were performed on ice. HBSS containing 0.3% BSA and 0.3 mM EDTA was used as a buffer for all incubations. DAPI was added to cells immediately prior to sort to distinguish live and dead cells. For all in vitro target cell engulfment assays, live macrophages (murine peritoneal macrophages: CD45+F4/80+DAPI-; murine BMDM: CD45+CD88+DAPI-; HMDM: CD45+HLA-DR+DAPI-) were separated into engulfing (TAMRA+) and non-engulfing (TAMRA-) populations. For in vivo IP ApoJ or IgGJ engulfment assays, live macrophages were identified as CD45+Ly6G-CD88+CD11b+F4/80hiDAPI- and TAMRA+ or TAMRA-. For bead engulfment assays, live macrophages (CD45+F4/80+DAPI-) were sorted into engulfing (Flash Red+) and non-engulfing (Flash Red-) populations. Liposomes had no label; liposome-fed macrophages were only sorted for viability. Primary antibodies used (source/clone): unlabeled CD16/32 (eBioscience/93), Ly6G (BD/IA8), F4/80 (ebioscience/BM8), CD45 (BD/30-F11), CD11b (eBioscience/M1/70), CD88 (BioLegend/20/70), CD64 (Biolegend/X54-5/7.1). All primary antibodies were used at a 1:400 dilution. All macrophages were sorted into 0.06% BSA-coated tubes on a Synergy cell sorter (Sony Biotech).

Treatment ID:

TR002089

Treatment Summary:

Apoptotic target cells Live Jurkat cells in RPMI-10 were exposed to 60,000 J of UV energy on a UV Stratalinker 2400 (Stratagene), then incubated at 37C for 4h to generate apoptotic Jurkat cells (ApoJ). Thymocytes, obtained by crushing WT murine thymi and filtering for a single cell suspension, were converted to apoptotic thymocytes in the same manner. For TAMRA-labeling, apoptotic targets were stained during the final hour by 15 minute incubation at 37C with 10M 5(6)-TAMRA,SE (Invitrogen) in PBS followed by two washes with RPMI-10 to remove excess TAMRA. Apoptotic targets were resuspended in fresh RPMI-10 and added to macrophages in culture at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2x106 ApoJ were injected IP. Other cell targets Jurkat cells destined to be IgGJ and NecJ were TAMRA-labeled prior to opsonization or killing using the procedure described above. Labeled cells were incubated for 30 minutes on ice with 25g/mL purified anti-human CD3 (Biolegend, Clone UCHT1) to generate IgGJ or incubated for 25 minutes at 55C to generate NecJ. All targets were resuspended in RPMI-10. IgG targets were added to macrophages at a ratio of 4:1 and co-cultured for 1h, NecJ targets were added to macrophages at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2x106 IgGJ were injected IP. Non-cell targets 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids Inc.) were used to generate PS-containing liposomes. Lipids suspended in chloroform were mixed at 70:30 POPC:POPS, then chloroform was evaporated under nitrogen. Dry lipids were resuspended in RPMI for a final concentration of 500M lipid and sonicated for 15 minutes until liposomes were formed and solution transitioned from opaque to clear. Liposomes were added to macrophages at a final concentration of 50M in RPMI-10. For other experiments, Flash Red 5m carboxylated polystyrene beads (PS/DVB-COOH·(660,690) carboxyl polystyrene beads, Bangs Laboratories Inc.) were added to macrophages at a final ratio of 1:2. Macrophages were co-cultured with non-cell targets for 1h and unengulfed targets were removed by washing macrophages three times with cold 1X PBS. Isotope-labeled metabolite use For some experiments, Jurkat T cells were cultured for 4 days in media containing [13C,15N]cell-free amino acid mixture (Sigma-Aldrich) (RPMI-1640 medium for SILAC, 10% v/v dyalized FBS, 1X Pen/Strep, 1mM HEPES, 4% v/v amino acid mixture). For some experiments, macrophages were cultured for 7 hours in media containing [13C,15N]Arginine (Cambridge Isotope Laboratories): RPMI-1640 medium for SILAC, 10% dyalized FBS v/v, 1X Pen/Strep, 1mM HEPES, 100M Sodium Pyruvate, 0.2 g/L [13C,15N]Arginine. FACS sorting engulfing and non-engulfing cells Single cell suspensions were incubated with unlabeled CD16/CD32 for 5 min to block non-specific FcR-mediated binding. Cells were stained with surface antibody panels for 20 min, and then washed. Cells were protected from light and incubations were performed on ice. HBSS containing 0.3% BSA and 0.3 mM EDTA was used as a buffer for all incubations. DAPI was added to cells immediately prior to sort to distinguish live and dead cells. For all in vitro target cell engulfment assays, live macrophages (murine peritoneal macrophages: CD45+F4/80+DAPI-; murine BMDM: CD45+CD88+DAPI-; HMDM: CD45+HLA-DR+DAPI-) were separated into engulfing (TAMRA+) and non-engulfing (TAMRA-) populations. For in vivo IP ApoJ or IgGJ engulfment assays, live macrophages were identified as CD45+Ly6G-CD88+CD11b+F4/80hiDAPI- and TAMRA+ or TAMRA-. For bead engulfment assays, live macrophages (CD45+F4/80+DAPI-) were sorted into engulfing (Flash Red+) and non-engulfing (Flash Red-) populations. Liposomes had no label; liposome-fed macrophages were only sorted for viability. Primary antibodies used (source/clone): unlabeled CD16/32 (eBioscience/93), Ly6G (BD/IA8), F4/80 (ebioscience/BM8), CD45 (BD/30-F11), CD11b (eBioscience/M1/70), CD88 (BioLegend/20/70), CD64 (Biolegend/X54-5/7.1). All primary antibodies were used at a 1:400 dilution. All macrophages were sorted into 0.06% BSA-coated tubes on a Synergy cell sorter (Sony Biotech).