Rat anti mouse cd16 cd32 antibody

PUER cells were preincubated with Rat anti-mouse CD16/CD32 antibody (BD Biosciences Cat# 553141, RRID:AB_394656) to reduce nonspecific binding. Cells were stained with (Phycoerythrin)-CF594-conjugated anti-F4/80 (T45-2342; BD Biosciences Cat# 565613, RRID:AB_2734770) or Biotin-conjugated anti-Gr-1 (RB6-8C5; BD Biosciences Cat# 553124, RRID:AB_394640) monoclonal antibodies from BD Biosciences. Gr-1 primary incubation was followed by incubation with Allophycocyanin-conjugated Streptavidin (BD Biosciences Cat# 554067, RRID:AB_10050396). Stained cells were analyzed on a BDFACSymphony flow cytometer (BD Biosciences). Data were analyzed using FlowJo (FlowJo, LLC). Positive fraction was estimated using FlowJo’s SE Dymax method.

The harvested immature BMDCs generated above were cultivated in 6-well plates in complete RPMI-1640 medium with stimulation of AES (10 µg/mL) or MES (10 µg/mL) at 37 °C, 5% CO₂ for 48–72 h. The same cell culture with 2 µg/mL LPS was used as a positive control and with bovine serum albumin (BSA) (10 µg/mL) (Thermo Fisher, Life Technologies) or PBS as a negative control. The stimulated BMDCs were washed and resuspended in 200 µL PBS containing 2 µL rat anti-mouse CD16/CD32 antibody (BD PharMingen, San Jose, CA, USA) for Fc receptors (FcR) block and then stained with P-phycoerythrin (PE)-labeled anti-mouse CD11c antibodies (BD PharMingen) and fluorescein isothiocyanate (FITC) anti-mouse CD80, CD86, CD40, or MHCII (BD PharMingen) for fluorescence-activated cell sorting (FACS) analysis. The cytokine levels in the BMDCs culture supernatants were determined by corresponding ELISA kits.

Cells were washed twice in phosphate buffered saline (PBS) and resuspended at 1 × 10⁶ cells/ml in FACS buffer (PBS/0.1% NaN₃/1% FBS). Cells were blocked with rat anti-mouse CD16/CD32 antibody (BD Biosciences) at 4°C for 5 min and then stained with fluorescein-conjugated anti-mouse SR-AI, PE-conjugated anti-mouse LOX1 (R&D Systems, Minneapolis, MN, USA), PE-conjugated anti-mouse CD36, FITC-conjugated CD11b, PE-conjugated anti-CD11b, PE-conjugated anti-mouse CD86, FITC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8a, FITC-conjugated anti-mouse CD19, and FITC-conjugated anti-mouse IA/IE (BD Biosciences) (all antibodies were diluted 1:100) for 30 min on ice in the dark. Matched isotype antibodies were used to show nonspecific binding. The cells were washed and resuspended in FACS buffer. A total of 10,000 events were acquired on a Navios flow cytometer (Beckman Coulter, La Brea, CA, USA), and the data were processed using Kaluza software (Beckman Coulter).

We analyzed 21 PDXs in the stromal study (S1 Table). The single-cell suspensions generated by tissue dissociation were incubated with rat anti-mouse CD16/CD32 antibody (BD Biosciences) at a concentration of 1 μg/10⁶ cells in 100 μl phosphate-buffered saline (PBS)/ 5% fetal calf serum (FCS), to block nonspecific binding. The cells were then stained with the appropriate monoclonal antibodies (listed in S3 Table). The stained cells were washed twice with PBS/FCS. We added 2 ng/ml DAPI (4',6'-diamidino-2-phenylindole, Invitrogen) to distinguish between live and dead cells. Data were acquired with a standard LSRII flow cytometer (BD Biosciences) equipped with 20 mW 488 nm, 20 mW 633 nm and 25 mW 406 nm lasers. We used the following filters for the measurement of fluorescence emission: 530/30 for FITC or AF488, 575/26 for PE, 610/20 for PE-TX Red, 660/20 for PE-Cy5, 695/40 for PerCP-Cy5.5 or PerCP-eFluor710, 780/60 for APC-Cy7, 660/30 for APC, 730/45 for AF700, 780/60 for APC-Cy7 and 450/50 for DAPI. Data were analyzed with FlowJo Software (Treestar, Ashland OR). For each model, we studied at least three tumors.

Flow cytometric analysis was performed as described previously (25 (link)). Briefly, tumor tissues were cut into pieces and digested with DMEM containing hyaluronidase (1.5 mg/ml, Sigma-Aldrich, USA), collagenase type 1A (1.5 mg/ml, Sigma-Aldrich, USA), and deoxyribonuclease I (20 U/ml, Sigma-Aldrich, USA) at 37°C for 45 min. The tissue mixture was passed through a 70-µm nylon cell strainer to make single cell suspension, which was then washed and resuspended in cold flow buffer (1% bovine serum albumin and 0.1% NaN₃ in PBS). After being blocked with a rat anti-mouse CD16/CD32 antibody (BD Pharmingen, USA), the following fluorochrome-conjugated anti-mouse antibodies were used: CD45-BV421, CD11b-BV510, CD8a-PE-Cy7, CD4-PE, CD25-APC, NK1.1-APC-Cy7, F4/80-FITC, CD206-PE-Cy7, CD11c-APC, and Gr-1-APC-Cy7 (all from BioLegend, USA). 7-Amino-actinomycin D (7AAD) (eBioscience, USA) was used as a viability dye to exclude dead cells. Flow cytometric data were collected using a Gallios flow cytometer (Beckman, USA) and analyzed using the Kaluza software (version 1.3).

pMø fixed with 2% PFD were washed three times with 0.1% PBS-Tween (Sigma-Aldrich) and incubated for 30 min on ice with 0.1% PBS-Tween20 containing purified rat anti-mouse CD16/CD32 antibody (BD Biosciences) at a dilution of 1:200 and 5% goat serum for p47^phox or 5% donkey serum for p22^phox staining, to block Fcγ receptors. After three washes with 0.1% PBS-Tween-20, the cells were incubated with α-p47^phox (H-195) or α-p22^phox (C-17) at a dilution of 1:100 in 0.1% PBS-Tween-20 containing 1% BSA. After 2 h of incubation, the cells were washed three times with 0.1% PBS-Tween-20 and incubated for 1 h in the dark with 0.1% PBS-Tween-20 containing 1% bovine serum albumin (BSA) and 1:1,000 dilution of Alexa Fluor^®488 goat anti-rabbit IgG (Invitrogen, by Thermo Fisher Scientific) for p47^phox or Alexa Fluor^®488 donkey anti-goat IgG (Invitrogen) for p22^phox staining. Slides were then washed twice with 0.1% PBS-Tween-20 followed by last wash with PBS. Finally, all the coverslips were mounted with DAPI-mounting medium (Vector Laboratories, Burlingame, CA, USA). Confocal microscopy was performed using an oil immersion 60× objective on the Nikon A1 microscope. The mean fluorescence intensity (MFI) was measured as the mean gray value of the maximum projection using ImageJ software.