Cy5 conjugated polyclonal donkey anti goat igg secondary

Organs and peripheral blood were obtained from euthanized adult hCD147Tg-NSG and WT-NSG mice. Organs were then mechanically and chemically digested with Collagenase IV (Gibco, 17104019) for 10 minutes using the gentleMACS Octo with heater (Miltenyi) before triturating through a 70μM cell strainer. The strained fraction was centrifuged at 400g for 5 minutes and then resuspended in ACK Lysis Buffer (Gibco, A1049201) for 5 minutes on ice. Phosphate-buffered saline (PBS) was added to quench the reaction and the cell suspension was centrifuged again. The supernatant was discarded, and the cell pellet was divided into various sample groups. Mouse cells were first preincubated with Fc Block according to manufacturer’s recommendations before proceeding to antibody incubation. Mouse CD147 was stained using primary goat anti-mouse CD147 (R&D Systems, BAF772) and visualized using Cy5-conjugated polyclonal donkey anti-goat IgG secondary (Jackson ImmunoResearch, 705–175-147). Human CD147 was stained using primary FITC-conjugated mouse anti-human CD147 antibody (Invitrogen, MEM-M6/1) Antibodies were applied at a 1:100 dilution per sample for 30 minutes on ice, rinsed with PBS, and resuspended in PBS. Acquisition was performed on a BD Accuri™ C6 Plus system and downstream analysis was done using FlowJo (Treestar).

Organs and peripheral blood were obtained from euthanized adult hCD147KI^het-NSG and WT-NSG mice. Organs were then mechanically and chemically digested with Collagenase IV (Gibco, 17,104,019) for 10 min using the gentleMACS Octo with heater (Miltenyi) before triturating through a 70 µm cell strainer. The strained fraction was centrifuged at 400 g for 5 min and then resuspended in ACK Lysis Buffer (Gibco, A1049201) for 5 min on ice. Phosphate-buffered saline (PBS) was added to quench the reaction and the cell suspension was centrifuged again. The supernatant was discarded, and the cell pellet was divided into various sample groups. Mouse cells were first preincubated with Fc Block according to manufacturer’s recommendations before proceeding to antibody incubation. Mouse CD147 was stained using primary goat anti-mouse CD147 (R&D Systems, BAF772) and visualized using Cy5-conjugated polyclonal donkey anti-goat IgG secondary (Jackson ImmunoResearch; 705–175-147). Human CD147 was stained using primary FITC-conjugated mouse anti-human CD147 antibody (Invitrogen, MEM-M6/1) Antibodies were applied at a 1:100 dilution per sample for 30 min on ice, rinsed with PBS, and resuspended in PBS. Acquisition was performed on a BD Accuri™ C6 Plus system and downstream analysis was done using FlowJo (Tree star).

Organs and peripheral blood were obtained from euthanized adult hCD147KI^het-NSG and WT-NSG mice. Organs were then mechanically and chemically digested with Collagenase IV (Gibco, 17104019) for 10 minutes using the gentleMACS Octo with heater (Miltenyi) before triturating through a 70 μm cell strainer. The strained fraction was centrifuged at 400g for 5 minutes and then resuspended in ACK Lysis Buffer (Gibco, A1049201) for 5 minutes on ice. Phosphate-buffered saline (PBS) was added to quench the reaction and the cell suspension was centrifuged again. The supernatant was discarded, and the cell pellet was divided into various sample groups. Mouse cells were first preincubated with Fc Block according to manufacturer’s recommendations before proceeding to antibody incubation. Mouse CD147 was stained using primary goat anti-mouse CD147 (R&D Systems, BAF772) and visualized using Cy5-conjugated polyclonal donkey anti-goat IgG secondary (Jackson ImmunoResearch; 705-175-147). Human CD147 was stained using primary FITC-conjugated mouse anti-human CD147 antibody (Invitrogen, MEM-M6/1) Antibodies were applied at a 1:100 dilution per sample for 30 minutes on ice, rinsed with PBS, and resuspended in PBS. Acquisition was performed on a BD Accuri^™ C6 Plus system and downstream analysis was done using FlowJo (Tree star).