Donkey anti rabbit bv421

Lymphocytes were isolated by passage through a 100 μm cell strainer (BD Biosciences, San Jose, CA). Single cell suspensions were subjected to red blood cell lysis by resuspension in ACK buffer. Following blocking of cell surface Fc receptors with anti-CD16/32, lymphocytes were surface stained using anti -CD4, -B220, -PD-1, -CXCR5. Cell viability was determined using Live/dead violet. Stromal cells were identified by staining with anti -CD45-PE, -CD31-PE-Cy7 in stain buffer (20 min, 4°C, Table 2). After extensive washing to remove unbound antibodies, cells were subjected to fixation and permeabilization using Fix/Perm reagent according to manufacturer’s instructions (BD Biosciences). Intracellular staining was performed using rabbit anti-β2AR (Abcam) [33 (link)–35 (link)] followed by donkey anti-rabbit BV421 (Biolegend, San Diego, CA) and goat anti-CXCL13 (RnD Systems, Minneapolis, MN) followed by donkey anti-goat Alex Fluor 647 (Invitrogen). After extensive washing to remove unbound antibodies, flow cytometry was performed on a LSRII running DIVA 6.0 (BD Biosciences). Data was analyzed using FlowJo v10.0 (Treestar, Ashland, Oregon).

Primary antibodies used in this study include ARL13b (Proteintech catalog 17711-I-AP), acetylated tubulin (Sigma catalog T6793), IFT88 (Thermo Fisher Scientific catalog PA5-18467), Inversin (Proteintech catalog 10585-I-AP), Dynein (Thermo Fisher Scientific catalog MA1-070), gTubulin (GeneTex catalog GTX113286), Alk1 (Abcam catalog ab51870), KLF4 (Proteintech catalog 11880-I-AP), HO-1 (BD, catalog 566391), NRF2 (BioLegend, catalog 939202), and bactin (Sigma catalog A5441 and Cell Signaling Technology catalog 4970P). Secondary antibodies used are goat anti-mouse PECy7 (BioLegend), donkey anti-rabbit PE (Thermo Fisher Scientific), donkey anti-goat AF657 (Thermo Fisher Scientific), donkey anti-rabbit BV421 (BioLegend), and donkey anti-rabbit AF488 (Thermo Fisher Scientific).