Cd3 pe dazzle

For patients with biopsies spanning multiple regions, descending colon and sigmoid-rectum cell suspensions were combined in equal numbers to provide an aggregate analysis of the lower colon. Cells stained with viability dye (0.4–0.6 × 10⁶) were resuspended in 100 μL of FACS wash buffer (FWB; PBS, 2%FBS, 1 mM EDTA) with the addition of the following antibodies: TruStain FcX (Biolegend 422302), CD3-PE/Dazzle (Biolegend 300450), CD45-Pacific Blue (Biolegend 304022), CD66b-PE/Cy7 (Biolegend 305116) and CD15-APC (Biolegend 301908). Cells were incubated on ice for 20 minutes, washed with 1 mL FWB, and then resuspended in 500 μL FWB for sorting on a FACS Aria II or III (BD Biosciences). For the majority of samples, 12–15 × 10³ live CD45⁺CD66b⁻ mononuclear cells (MNC) cells and CD3⁺ cells (pre-gated on live MNC) were separately sorted into T cell media (RMPI + 10% FBS) and immediately transferred to ice. CD66b+ granulocytes were excluded from the analysis because these cells interfere with scRNA-seq using the 10X Genomics protocol.

LPMC were cultured for 4 days in the presence or absence of butyrate (0.25 and 0.5 mM). After four days of culture, cells were stained using FOXP3 Fix/Perm Buffer Set (Biolegend) with the following antibodies: CD45 (PerCpCy5.5, eBiosciences, Clone: 2d1), CD3 (PE-Dazzle, Biolegend, Clone: UCHT1), CD4 (APC, TONBO, Clone: RPA-T4),CD8 (PE-Cy7, TONBO, Clone: RPA-T8), CD25 (PE, BD, Clone: M-A251) and FOXP3 (AF488, eBiosciences, Clone: PCH101) and rat IgG2a (eBiosciences).