Early in life, a combination of environmental insults and genetic pre-disposition results in an autoimmune reaction to pancreatic b-cells in the Islets of Langerhans, leading to b-cell destruction and a lack of insulin production. Over time, near-complete loss of insulin production leads to Type I Diabetes (T1D), an often-deadly disease that requires lifetime treatment. Many studies have focused on immune system components that drive T1D autoimmunity, particularly the immune cells that infiltrate the islets of Langerhans. However, acute insulitis is extremely difficult to detect, as T1D pancreas samples are rare and individual islet destruction is thought to happen rapidly and sporadically. Therefore, studies have focused on autoimmune imprints within memory T and B cells that may develop after insulitis. Memory T and B cells that target b-cells would likely reside in lymph nodes that drain the pancreas and are potentially detectable in circulation or in the spleen. The Human Pancreas Analysis Program has organized a protocol to isolate single immune cells from the blood, spleen, and peri-pancreatic lymph nodes (PLNs) from healthy, pre-diabetic, and T1D patients. Herein, we describe the standardized protocol of single cell isolation from blood, spleen, PLNs, mesenteric LNs, and superior mesenteric LNs.