Easy sep cd4 isolation kit

CD4+ T cells were isolated from the spleen and lymph nodes (brachial, axial, inguinal and mesenteric) of CD45.2 Pak2^+/+;Foxp3-Cre (WT) and Pak2^F/F;Foxp3-Cre (KO) mice by magnetic depletion using the Easy Sep CD4 Isolation Kit (Stemcell Technologies). Using these cells, we sorted WT and Pak2-deficient CD4+YFP+ Tregs. Simultaneously, CD4+CD25−CD45RB^hi naïve T cells were sorted from WT CD45.1 mice. Naïve T cells were stained with 5 μM carboxyfluorescein succinimidyl ester (CFSE) for 8 minutes at room temperature. In combination with 1x10⁵ irradiated splenocytes and 1 μg/ml anti-CD3, 4x10⁴ CD4+CD25−CD45RB^hi naïve T cells were incubated with varying ratios of CD4+YFP+ Tregs derived from WT or KO mice (Ratio of naïve:Treg; 1:0, 1:1, 1:2, 1:4, 1:8, 1:16, 1:32) in complete mouse media. Cells were incubated for 72 hours at 37 °C prior to harvesting. To separate out CFSE-stained T cells from unstained APCs and Tregs, cells were stained with anti-CD45.1 and CFSE dilution was monitored by flow cytometry.

CD8⁺ T cells were isolated from the spleen and s.c. LNs (axillary, brachial, inguinal, popliteal, cervical) of OTI mice using the EasySep CD8⁺ isolation kit (Stemcell 19853). Similarly, CD4⁺ T cells were isolated from the spleen and s.c. LNs of OTII mice using the EasySep CD4⁺ isolation kit (Stemcell 19852). Spleens were first mashed into a single cell suspension and lysed with ACK lysis buffer (Gibco A1049201). LNs were digested with 1 mg/mL Ca²⁺ supplemented Collagenase D (Roche 11088866001) for 45 min at 37°C and gently mashed into a single cell suspension. Suspensions from the LNs and spleen were pooled and subjected to magnetic cell isolation using the kits. The OTI and OTII cells were labeled with 1 μM CFSE (eBioScience 65-0850-84) for 6 min at RT, washed with sterile PBS buffer, quantified and resuspended in saline buffer for injection. 5x10⁵ - 1x10⁶ cells of each OTI and OTII cells were injected into mice via i.v. tail vein injection.

CD4⁺B220^–Tetramer⁺ (Tet⁺) or Tetramer⁻ (Tconv) cells were sorted from pMHCII-NP-treated mice by flow cytometry. TF-like cells within the tetramer⁻ CD4⁺ gate were identified by staining with anti-CXCR5 and anti-PD-1 mAbs. Briefly, CD4⁺ T cells were enriched from spleen cell suspensions using an EasySep CD4 Isolation Kit (STEMCELL Technologies, Vancouver, BC), stained with pMHCII tetramers and mAbs and sorted into the tetramer⁺ CD4⁺ and tetramer⁻ CD4⁺CXCR5^hiPD-1^hi subsets by flow cytometry, as described above. The purities of TF and tetramer⁺ cells were 85.8 ± 4.4% and 96.5 ± 1.5% (mean ± SE, n = 4), respectively. FACS-sorted cells (10⁵) were challenged with anti-CD3/anti-CD28 mAb-coated beads (Dynabeads T-cell Activator, ThermoFisher, Waltham, MA) for 48 h. Cytokine contents in the supernatants were measured via a Multiplexing LASER Bead Assay (Eve Technologies, AB, Canada). Total RNA was prepared for RNAseq using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany).
TFH cells (PD-1^hiCXCR5^hi) were generated by immunizing NOD mice intraperitoneally with KLH (keyhole limpet hemocyanin) or KLH-DNP (Sigma‒Aldrich, St. Louis, MO, USA) once a week for three consecutive weeks for a total of 3 times (100 µg/dose, CFA + IFA + IFA).