Apc rat igg2a

Single cells digested from skin wounds were pre-incubated with purified anti-CD16/CD32 antibody (101301, BioLegend) (1.0 μg per 10⁶ cells in 100 μl volume) for 5 to 10 min to block Fc receptors. The cell suspensions were then co-incubated with fixable viability dye (eFluor™ 780, 65-0865-14, eBioscience) and antibodies against surface markers CD45 (PE/Cyanine7, 147703, BioLegend), CD3 (PE, 100205, BioLegend), and F4/80 (FITC, 123107, BioLegend) at 1:400 dilution for 30 min at 4 °C in the dark (100 μl per antibody per sample). After fixation and permeabilization, cells were incubated with antibodies against intracellular marker CD68 (APC, 137007, Biolegend) at 1:400 dilution for 20 min at 4 °C in the dark (100 μl per antibody per sample). Isotype controls of CD45 (PE/Cyanine7 Rat IgG2b, κ, 400617, Biolegend), CD3 (PE Rat IgG2b, κ, 400607, Biolegend), F4/80 (FITC Rat IgG2a, κ, 400505, BioLegend) and CD68 (APC Rat IgG2a, κ, 400511, Biolegend) were used in same concentration. Flow cytometry analysis was performed using Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo (v10.8.1). The experiments were performed three times independently (n = 3).

The following antibodies of cell surface molecules were used: Allophycocyanin (APC) anti-mouse CD45 (Thermo Fisher Scientific Cat# MCD4505, RRID:AB_10376146), anti-mouse CD31 (BioLegend Cat# 102410, RRID:AB_312905), anti-mouse CD166 (Thermo Fisher Scientific Cat# 17-1661-82, RRID:AB_2573170), anti-mouse CD200 (BioLegend Cat# 123810, RRID:AB_10900447), anti-mouse SCA1 (BioLegend Cat# 122511, RRID:AB_756196), anti-mouse CD105 (BioLegend Cat# 120414, RRID:AB_2277914); APC Rat IgG2a, κ isotype control antibody (BioLegend Cat# 400512, RRID:AB_2814702), APC Rat IgG2b, κ isotype control antibody (BioLegend Cat# 400612, RRID:AB_326556), APC Rat IgG1, κ isotype control (BioLegend Cat# 400412, RRID:AB_326518); Alexa Fluor 647 anti-mouse CD9 (BioLegend Cat# 124810, RRID:AB_2076037), Alexa Fluor 647 Rat IgG2a, κ isotype control antibody (BioLegend Cat# 400526, RRID:AB_2864284).
Cells (n=3 with cells from three different mice) were stained with antibodies or IgG isotype controls for 30  min at room temperature. Stained cells were analyzed on a FACSCalibur or BD LSR II flow cytometer (BD Biosciences). Positive cells were gated based on both unstained and isotype-matched IgG-stained cells. Data analysis was performed using FlowJo software (BD Biosciences).

RAW264.7 cells in the logarithmic growth phase were spread to a 6-well plate at 10⁶ cells/well. After at least 6 h, the cells were completely attached to the wall, and the lysate and control were added separately. After 24 h of treatment, RAW264.7 cells were stained with FITC anti-mouse F4/80 (clone: FJK-16s, Invitrogen, Waltham, Massachusetts), APC anti-mouse CD86 (clone: GL-1, Biolegend, San Diego, CA) and PE anti-mouse CD206 (clone: MR6F3, Invitrogen, Waltham, Massachusetts) according to the protocol of the antibodies (M1 macrophage: F4/80+CD86+, M2 macrophage: F4/80+CD206+) and subjected to flow cytometry (LSR II, BD). Detection of SIRPα on macrophages in spleen from mouse used FITC anti-mouse F4/80, APC anti-mouse CD11b (clone: M1/70, Biolegend, San Diego, CA), and PE anti-mouse SIRPα (clone: P84, Biolegend, San Diego, CA). All flow cytometry used to detect macrophage typing in this study was set up with an antibody isotype control group (PE Rat IgG2a, clone: RTK2758, Biolegend, USA; APC Rat IgG2a, clone: RTK2758, Biolegend, USA; FITC Rat IgG2a, clone: RTK2758, Biolegend, USA).

Tumor tissues were harvested in RPMI Medium 1640 with 10% heat-inactivated FBS, 1% penicillin and streptomycin (all from HyClone™). Tumors were minced and incubated for 30 min in DPBS (Well-gene) with 0.5 mg/ml Collagenase IV (Sigma) at 37 °C. Tissues were squeezed through a 100 μm cell strainer (# 93100, SPL), re-filtered through a 70 μm cell strainer (# 93070, SPL) subjected for 5 min to Red Blood Cell Lysis Buffer (#10-548E, Lonza). Flow cytometry was performed to determine cell surface antigen expression by 30-min incubation on ice with pertinent antibodies. The following monoclonal antibodies were used: mouse-specific monoclonal antibodies used were PerCP/Cyanine5.5 anti-mouse CD45 (#103131, BioLegend), APC-anti-mouse CD8a (#100712, BioLegend) and corresponding isotype control mAbs PerCP/Cy5.5 Rat IgG2b (#400632, BioLegend) and APC Rat IgG2a (#400512, BioLegend). The cells were washed thrice with PBS containing 2% FBS and fixed in suspension with 4% paraformaldehyde and stored at 4 °C until analysis with a CytoFLEX flow cell counter (Beckman). We analyzed the data using FlowJo software.

Single cells digested from skin wounds were pre-incubated with purified anti-CD16/CD32 antibody (101301, BioLegend) (1.0 μg per 10⁶ cells in 100 μl volume) for 5 to 10 min to block Fc receptors. The cell suspensions were then co-incubated with fixable viability dye (eFluor™ 780, 65-0865-14, eBioscience) and antibodies against surface markers CD45 (PE/Cyanine7, 147703, BioLegend), CD3 (PE, 100205, BioLegend), and F4/80 (FITC, 123107, BioLegend) at 1:400 dilution for 30 min at 4 °C in the dark (100 μl per antibody per sample). After fixation and permeabilization, cells were incubated with antibodies against intracellular marker CD68 (APC, 137007, Biolegend) at 1:400 dilution for 20 min at 4 °C in the dark (100 μl per antibody per sample). Isotype controls of CD45 (PE/Cyanine7 Rat IgG2b, κ, 400617, Biolegend), CD3 (PE Rat IgG2b, κ, 400607, Biolegend), F4/80 (FITC Rat IgG2a, κ, 400505, BioLegend) and CD68 (APC Rat IgG2a, κ, 400511, Biolegend) were used in same concentration. Flow cytometry analysis was performed using Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo (v10.8.1). The experiments were performed three times independently (n = 3).

The immunological activity of IFN-γ expressed by URB was evaluated by macrophages activation. RAW 264.7 macrophages were seeded into a 96-well plate (8 × 10³ cells per well), and then incubated in 100 μl DMEM medium containing 10% FBS for 12 h. The bacterial culture supernatant with corresponding concentrations of IFN-γ (50–150 pg/ml) was added into the macrophages and incubated for 48 h. To detect the phenotype of macrophages, the treated RAW 264.7 macrophages were stained with corresponding antibodies: CD86-PE (Biolegend, Cat No. 105007, dilution ratio 1:100), CD80-BV421 (Biolegend, Cat No. 104725, dilution ratio 1:100) and CD206-APC (Biolegend, Cat No. 141707, dilution ratio 1:100) according to the manufacturer’s instructions and analyzed by flow cytometry. PE-Rat IgG2a (Biolegend, Cat No. 400507, dilution ratio 1:100), BV421-IgG (Biolegend, Cat No. 400935, dilution ratio 1:100) and APC-Rat IgG2a (Biolegend, Cat No. 400511, dilution ratio 1:100) were used for isotype control. After that, The NO detection kit (Beyotime, Cat No. S0021S) was used to detect the NO level in macrophage culture medium. Moreover, RAW 264.7 macrophages treated with different levels of IFN-γ were collected and used to co-incubate with tumor cells at a MOI ratio of 5:1 in 96-well plate for 12 h, and the tumor killing activity was evaluated with CCK-8 kit.

Single cells digested from skin wounds were pre-incubated with purified anti-CD16/CD32 antibody (101301, BioLegend) (1.0 μg per 10⁶ cells in 100 μl volume) for 5 to 10 min to block Fc receptors. The cell suspensions were then co-incubated with fixable viability dye (eFluor™ 780, 65-0865-14, eBioscience) and antibodies against surface markers CD45 (PE/Cyanine7, 147703, BioLegend), CD3 (PE, 100205, BioLegend), and F4/80 (FITC, 123107, BioLegend) at 1:400 dilution for 30 min at 4 °C in the dark (100 μl per antibody per sample). After fixation and permeabilization, cells were incubated with antibodies against intracellular marker CD68 (APC, 137007, Biolegend) at 1:400 dilution for 20 min at 4 °C in the dark (100 μl per antibody per sample). Isotype controls of CD45 (PE/Cyanine7 Rat IgG2b, κ, 400617, Biolegend), CD3 (PE Rat IgG2b, κ, 400607, Biolegend), F4/80 (FITC Rat IgG2a, κ, 400505, BioLegend) and CD68 (APC Rat IgG2a, κ, 400511, Biolegend) were used in same concentration. Flow cytometry analysis was performed using Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo (v10.8.1). The experiments were performed three times independently (n = 3).