TITLE: HPAP Processing Protocol
AUTHORS: Michael Betts, Gregory Golden

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div></div>

[STEPS]
?. BLOOD1. Spin down vials at 2000rpm for 15min at room temperature2. Aliquot plasma into cryovials and freeze and store at  ; toss remaining plasma3.  Measure volume of spun down blood and transfer to a new conical tube4. Using a 1:1 ratio of spun blood to R10 media, wash vials and transfer to the conical with the spun down blooda. R10 media = RPMI + 10% fetal bovine serum + 2mM L-Glutamine + 100 U/mL Penicillin + 100 μg/mL Streptomycin 5. In a SepMate™-50 conical, add  of ficoll below the physical barrier6. Slowly add the blood to the top of the ficoll, taking care not to mix the two7. Spin down at for 10min at room temperature8. Once separated, pour the supernatant with the PBMCs into a fresh conical9. Spin down at  for 7min10. Pour off supernatant and resuspend the pellet in ~2mL ACK Lysis buffer11. Incubate at room temperature for 5min and then quench with  R10 media12. Spin down at for 7min13. Resuspend pellet in  of media14. Count cells15. Spin down at for 7min16. Pour off supernatant and resuspend the pellet in FBS + 10% DMSO at 5-10 million cells per mL17. Freeze in aliquots at  in a Mr. Frosty freezing container for 24-48hrs18. For long term storage, store in liquid nitrogen
-80 °C
15 mL
1200 g
500 g
18 mL
500 g
10 mL
500 g
1 mL
-80 °C
?. LYMPH NODE PROCESSING1. Prepare media by adding  DNase +  R10 media into one conical for each tissue type2. Weigh tissue in a culture dish and record mass3. Add  of prepared media to the culture dish 4. Place tissue in dish and gently cut away the fat 5. Rinse with  of prepared media 6. For histology:    a. Cut off a small piece of tissue (~1-2cm) and place in freshly prepared PBS + 4% PFA solution    b. Write date and time on the tube and store for ~24hrs at RT    c. Transfer to conical with 80% EtOH and store at 7. Place tissue pieces in a 70μm cell strainer and grind the tissue with the top end of a   syringe 8.  Strain media in plate through the cell strainer into a fresh  conical 9.  Rinse plate several times with remaining media and strain through the cell strainer into the conical 10. Spin down at for 7min11. Resuspend pellet in  of R10 media 12. Count cells13. Spin down at for 7min14. Pour off supernatant and resuspend the pellet in FBS + 10% DMSO at 5-10 million cells per mL15. Freeze in aliquots at  in a Mr. Frosty freezing container for 24-48hrs16. For long term storage, store in liquid nitrogen
50 µl
50 mL
10 mL
10 mL
50 mL
50 mL
4 °C
5 mL
50 mL
500 g
10 mL
500 g
1 mL
-80 °C
?. SPLEEN PROCESSING1. Prepare media by adding  DNase + Collagenase + R10 media2. Weigh tissue in a culture dish and record mass3. Cut tissue into ~2cm pieces4. Dissociate using a gentleMACS with the tissue suspended in prepared media5. Incubate for 15min at  on a rotator6. Repeat gentleMACS dissociation7. Strain suspension through a 100μM filter 8. Spin down at for 10min9. Pour off supernatant and mix in ~10mL ACK Lysis buffer 10. Let sit for 5min and then quench with of R10 media 11. Strain suspension through a 70μM filter 12. Spin down vials at  for 10min at room temperature13. Add  ficoll to a clean  conical 14. Resuspend cells and very carefully add to the top of the ficoll15. Spin down at 2200rpm for 20min with the brake off 16. Resuspend cells with of R10 media 17. Dilute  of the suspension in  of PBS18. Count cells and multiply by 1000 to obtain the final cell count19. Spin down at  for 10min20. Pour off supernatant and resuspend the pellet in 20-30mL FBS + 10% DMSO21. Freeze in 20-30 aliquots at in a Mr. Frosty freezing container for 24-48hrs22. For long term storage, store in liquid nitrogen
50 µl
50 µl
50 mL
37 °C
500 g
40 mL
500 g
10 mL
50 mL
10 mL
10 µl
990 µl
500 g
-80 °C