Percp cy5.5 anti cd3

Mouse colons were washed in cold CMF-HBSS and placed in CMF-HBSS containing 5 mM EDTA and shaken gently at 37 °C for 30 min to remove epithelial cells. The remaining colon tissue was minced and agitated in RMPI with 2 mg/mL dispase (Gibco), 0.5 mg/mL collagenase I (Gibco) and 0.2 mg/mL DNase (Roche) at 37 °C for 45 min. Tissue was broken up using a 19-gauge needle and filtered through a 100 μM filter. Cells were centrifuged at 350 × g for 20 min and used for flow cytometry. For cell sorting, cells were resuspended in 40%Percoll/RPMI and spun at room temperature for 20 min at 600 × g and the cell pellet was collected. Single-cell suspensions were washed with FACS buffer (PBS/1% FCS) and incubated with combinations of the following Abs: APC-Cy7 anti-CD45 (Clone 30-F11), Pacific Blue anti-CD90 (Clone 30.H12), APC anti-EpCam (Clone G8.8), Alexa Fluor 594 anti-vimentin (Clone W1622A), APC anti-CD140a (Clone APA5), Alexa Fluor 647 anti-CD74 (Clone In1/CD74), PE anti-CD49a (Clone HMα1), PE Cy7 anti-F4/80 (Clone BM8) and PerCP Cy5.5 anti-CD3 (Clone 17A2) (Biolegend, San Diego, CA). Cells were then analyzed with an LSR Fortessa cytometer (BD Biosciences, San Jose, CA). Citrine⁺ cells were sorted with a Sony SH800S supported by NIH S10OD023410.

Whole blood was incubated with 25% normal mouse serum (Jackson ImmunoResearch, 015–000‐120) to block non‐specific sites. After 5–10 min, primary antibody was added at 1:25 dilution: FITC anti‐CD16 (Biolegend, 302006), PE anti‐CD14 (Tonbo, 50‐0149‐T100), PerCPCy5.5 anti‐CD3 (Biolegend, 300328), APC anti‐HLADR (Biolegend, 307610), and AlexaFluor700 anti‐CD66b (Biolegend, 305114). Samples were incubated at room temperature for 20 min protected from light and then incubated with 1 mL of VersaLyse (Beckman Coulter, A09777) at room temperature for 20 min to remove red blood cells. Tubes were centrifuged at 400g for 3 min at room temperature, supernatant was aspirated, and cells were resuspended in 1 mL of room temperature PBS containing 1% BSA (SeraCare, 1900‐0016). Tubes were centrifuged again at 400g for 3 min, supernatant was aspirated, and cells were resuspended in 300 μL of ice‐cold freshly prepared 0.2% paraformaldehyde (Sigma, 158127) prepared in PBS.

The cells were incubated with Fc blockade reagent for 20 min at room temperature and stained with fluorochrome-conjugated antibodies in serum and sodium azide-containing buffer. Flow cytometry was performed as previously described (16 (link)). Antibodies in the present study were listed as follows: FITC anti-CD56 (Cat#362546, BioLegend, USA), PE anti-NKp30 (Cat#325208, BioLegend, USA), anti-CD56 (Cat#362508, BioLegend, USA), anti-NKp44 (Cat#325108, BioLegend, USA), anti-CD27 (Cat#356406, BioLegend, USA), PerCP-Cy5.5 anti-CD3 (Cat#317336, BioLegend, USA), anti-NKG2D (Cat#320818, BioLegend, USA), APC anti-NKp46 (Cat#331918, BioLegend, USA), anti-CD11b (Cat#301310, BioLegend, USA), anti-CXCR6 (Cat#356006, BioLegend, USA), anti-CD3 (Cat#317318, BioLegend, USA), PE-Cy7 anti-NKp46 (Cat#331916, BioLegend, USA), anti-CD3 (Cat#317334, BioLegend, USA), APC-Cy7 anti-CD3 (Cat#317342, BioLegend, USA), and anti-CD45 (Cat#304014, BioLegend, USA). FlowJo software was used for data analyzing (Tree Star, Inc., Ashland).