TITLE: Protocol to isolate, cryopreserve, and fix  mouse PBMCs for IGVF
AUTHORS: Elisabeth Rebboah

[DESCRIPTION]
This protocol describes isolation of peripheral blood mononuclear cells (PBMCs, tissue ID: 20) from whole blood of 10 week old mice, cryopreservation, thawing, and fixation for single-cell RNA-seq using the Parse Biosciences protocol (Split-seq). For 8-10 samples, PBMC isolation and cryopreservation take about 3 hours from start to finish, while thawing and fixation take around 1 hour.

The final result is 1 aliquot of a fixed single-cell suspension for Parse Bio scRNA-seq ("Split-seq") for each sample at >= 2,500 cells/ul stored at -80C. The intermediate result is approximately 1 million cryopreserved PBMCs per sample in 1 mL 10% DMSO + FBS, stored in liquid nitrogen until thawing and fixing. 

The first part of the protocol describes isolation and cryopreservation. Due to low input volumes (ranging from 100-1,000 ul), whole blood is diluted 1:2 in PBS and Ficoll-Paque density gradient centrifugation is performed in 2 mL tubes or blood collection tubes. Buffy coat is collected and red blood cells are lysed before counting on a hemocytometer. Plasma is also collected and stored in 200 ul aliquots at -20C. PBMCs are cryopreserved in liquid nitrogen in 10% DMSO FBS. Cryopreserved PBMCs are suitable for fixation for a variety of downstream protocols including single-cell RNA-seq, but not cell culture. If PBMCs are meant to be grown after isolation, perform all steps after the initial blood collection in a cell culture hood / biosafety cabinet with sterile technique. The second part of the protocol describes thawing and fixation. Cells are gently thawed and washed with culture media and PBS. Due to low cell recovery, we modify the original Parse Biosciences Evercode fixation protocol (attached) by using half volumes of all fixation reagents.

[STEPS]
SECTION: Setup
1. Set centrifuge to 19C.
SECTION: Setup
2. Prepare 1 ice bucket.
SECTION: Setup
3. Thaw and filter FBS with a cell culture filter unit and aliquot into 15 mL conical tubes. 
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4. Prepare PBS-EDTA by adding EDTA powder to PBS and filter with a cell culture filter unit. 500 mL of PBS-EDTA should be enough for around 16 samples. Store at room temperature, or 4C if not using that week.
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5. Prepare PBMC-RSB and 1x RBC lysis buffer at room temperature.
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6. Prepare 20% DMSO FBS on ice.
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7. Distribute 20 ul dye into 10 PCR tubes for cell counting.
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8. Optional: Prepare 1% BSA-DEPC stock tubes by adding BSA powder to 100 mL DEPC. Coat 5 mL tubes by adding 5 mL 1% BSA-DEPC in the cell culture hood (biosafety cabinet), incubating for 30 minutes, emptying the tubes, and drying for 30 more minutes. More detailed instructions are posted on the cell culture hood. We typically prepare many tubes ahead of time and store at 4C.
SECTION: PBMC isolation and cryopreservation
9. Collect blood in labeled EDTA-coated tubes at the vivarium during dissection. Cap and invert tubes so that all the blood touches the walls of the EDTA-coated tubes to prevent clotting and keep tube on rotating platform as other dissections are underway. 
SECTION: PBMC isolation and cryopreservation
10. At the lab, briefly spin tubes to collect any remaining blood from the walls/cap.
SECTION: PBMC isolation and cryopreservation
11. Measure blood volume with a 1 mL pipette and record.
SECTION: PBMC isolation and cryopreservation
12. Dilute blood 1:2 with PBS. E.g. 900 ul blood + 900 ul PBS.
SECTION: PBMC isolation and cryopreservation
13. Aliquot appropriate amount of Ficoll-Paque density gradient media. 
Multiple final diluted blood volume by 0.75x. E.g. 1800 u diluted bloodl x 0.75 = 1350 ul Ficoll-Paque. Use the smallest possible tube. If total blood volume is >500 ul, use another EDTA-coated blood collection tube. If total blood volume is <500 ul, use a 2 mL tube. 
SECTION: PBMC isolation and cryopreservation
14. Layer diluted blood on top of Ficoll-Paque.
SECTION: PBMC isolation and cryopreservation
15. Centrifuge 400g for 30 minutes at 19°C with no brake. Be careful to balance the centrifuge.
SECTION: PBMC isolation and cryopreservation
16. Collect plasma and distribute in PCR tubes, 200 ul each. Label and store in IGVF Plasma box at -20C.
SECTION: PBMC isolation and cryopreservation
17. Collect PBMC layer in ~1 mL into a labeled 5 mL tube (1% BSA-coating optional). Fill tube to 5 mL with PBMC-RSB to wash cells and remove Ficoll-Paque. Discard gradient tube with remaining Ficoll-Paque and red blood cells in biohazard trash.
SECTION: PBMC isolation and cryopreservation
18. Centrifuge 400g at 19°C for 8 minutes with full brake.
SECTION: PBMC isolation and cryopreservation
19. Remove supernatant until 100 uL PBMC-RSB is remaining in the tube. 
SECTION: PBMC isolation and cryopreservation
20. Add 1 mL 1x RBC lysis buffer to 100 uL cells. Vortex cells at low speed for 5 seconds and incubate for 10 minutes at room temperature.
SECTION: PBMC isolation and cryopreservation
21. Centrifuge 400g at room temperature for 8 minutes with full brake.
SECTION: PBMC isolation and cryopreservation
22. Remove supernatant and resuspend cells in 500 uL PBMC-RSB for counting. 
Do not count cells later when they’re in FBS, the proteins in the FBS cause background autofluorescence under the microscope.
SECTION: Counting
23. Aliquot 20 ul from each 5 mL tube containing 500 uL cells into the PCR tubes containing 20 ul dye.
SECTION: Counting
24. Label each side of a disposable hemocytometer with the sample number.
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25. At an angle, pipette 10 ul of cells in dye into the divots on the slide. Use 1 side per sample.
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26. Under EVOS FL Auto 2 microscope at 10x resolution (PBMCs are small), count cells in 2 squares on opposite sides of the grid. Use the DAPI channel to tell PBMCs from red blood cells or debris.
SECTION: Counting
27. Sum up squares (accounts for the 1:2 dilution), and convert to cells/mL by multiplying by 10^4. E.g. (45 + 63)*10^4= 1,080,000 cells per mL.
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28. Take pictures on the microscope for your records.
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29. Centrifuge for the last time at 400g at room temperature for 8 minutes with full brake.
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30. Remove supernatant and resuspend cells in 500 uL cold FBS. Move the cells to labeled cryotubes.
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31. Slowly add 500 uL 20% DMSO in FBS for a final volume of 1 mL. Mix gently after all 500 uL are added. Keep cryotubes on ice. Final concentration is 10% DMSO in FBS.
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32. Move tubes to Mr. Frosty and to the -80C freezer.
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33. The next day, move cryotubes to liquid nitrogen racks and record location.
37. Add 50 μL of RNase Inhibitor directly into the Cell Prefixation Buffer tube. Mix thoroughly by pipetting up and down 5x with a P1000 set to 750 μL. To record the addition of RNase Inhibitor, mark the cap of the Cell Prefixation Buffer tube, and store on ice.
SECTION: Cell Fixation
49. Pipette cells through a 40 μm strainer into a new 15 mL polypropylene centrifuge tube with a P1000 and store on ice.
Critical! To ensure that all of the liquid passes through the strainer, press the tip of the pipette against the filter and steadily depress down the pipette plunger. All of the liquid should pass through the strainer in ~1 second.
SECTION: Thawing cryopreserved PBMCs
42. Hold the lower half of the vials in a 37°C water bath for 30–45 sec, flicking intermittently. 
Note: Do not submerge the vial completely under water. Do not thaw completely. Allow about 20% of the ice crystals to remain intact.
SECTION: Thawing cryopreserved PBMCs
43. While 20% of the ice crystal is still visible, take the vials out of the water bath and wipe them with 70% ethanol.
SECTION: Thawing cryopreserved PBMCs
44. Transfer the cell suspension immediately from the cryovial to the 15 mL tube containing culture medium using a P1000 pipette. Note: Dispense the culture media dropwise from the 15 mL tube to the cryovial to slowly thaw the ice crystals. Aseptic technique not necessary since we are proceeding directly to fixation.
SECTION: Thawing cryopreserved PBMCs
41. Transport cryovials containing PBMCs from the freezer to the water bath on dry ice. We prepare 8 samples at a time.
SECTION: Thawing cryopreserved PBMCs
45. Transfer all the contents in the cryovial to the culture medium in the 15 mL tube. Use an additional 1 mL of culture medium to rinse out the cryovial and ensure maximum recovery of PBMCs.
SECTION: Thawing cryopreserved PBMCs
46. Gently invert the 15 mL polypropylene tube 3–4 times for even cell distribution and centrifuge at 350g for 5 min at room temperature (21°C).
SECTION: Thawing cryopreserved PBMCs
47. Carefully remove the supernatant. Leave a small amount of media at the bottom to re-suspend the pellet obtained after centrifugation.
SECTION: Thawing cryopreserved PBMCs
48. Gently loosen the cell pellet by flicking. Add 10 mL of culture medium. Repeat steps 41 and 42 in this section with PBS rather than culture media to wash off media. Resuspend cells in 375 ul Cell Prefixation Buffer + BSA with a P1000.
SECTION: Thawing and fixation setup
34. Prepare complete medium by adding 20% FBS and 1% penicillin-streptomycin to your media of choice (we use DMEM, but RPMI works too). Warm culture medium to 37°C.
SECTION: Thawing and fixation setup
35. Aliquot 10 mL of pre-warmed culture medium into a 15 mL polypropylene tube. 
SECTION: Thawing and fixation setup
36. Open 1 new Parse Biosciences Cell Fixation kit. Add 550 μL of Cell Fixation Additive directly into the Cell Fixation Solution. Mix thoroughly by pipetting up and down 5x with a P1000 set to 750 μL. To record the addition of Cell Fixation Additive, mark the cap of the Cell Fixation Solution tube, and store on ice.
38. Add 17 μL of RNase Inhibitor directly into the Cell Buffer tube. Mix thoroughly by pipetting up and down 5x with a P1000 set to 750 μL. To record the addition of RNase Inhibitor, mark the cap of the Cell Buffer tube, and store on ice.
39. Record today’s date on the Cell Fixation Reagents kit box. 
Note: After mixing reagents, Evercode Cell Fixation kits should only be freeze-thawed once and stored for up to 1 month at -20°C. Longer storage or additional freeze-thaws will compromise data quality.
40. Prepare Cell Prefixation Buffer + BSA. Add 200 μL of 7.5% BSA to 3 mL Cell Prefixation Buffer. Cell Prefixation Buffer + BSA should be prepared fresh and used the same day. Mix thoroughly by pipetting up and down 5x and store on ice.
SECTION: Cell Fixation
50. Add 125 μL of Cell Fixation Solution to the 15 mL tube and mix immediately by pipetting up and down exactly 3x with a P1000 set to 125 μL. Return the tube to ice.

Critical! Do NOT perform additional mixing at this step. Also, ensure the Cell Fixation Solution contains Cell Fixation Additive, as indicated by a mark on the tube cap.
SECTION: Cell Fixation
51. Incubate on ice for 10 minutes
SECTION: Cell Fixation
52. Add 40 μL of Cell Permeabilization Solution to the 15 mL tube and mix thoroughly by pipetting up and down 3x with a P1000 set to 125 μL. Return the tube to ice.
SECTION: Cell Fixation
53. Incubate on ice for 3 minutes.
Note: Do NOT vortex the Cell Neutralization Buffer. Prior to use, invert the tube 5x to mix.
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54. Add 2 mL of Cell Neutralization Buffer to the 15 mL tubes. Gently invert the 15 mL tube once to mix and return to ice.
SECTION: Cell Fixation
55. Centrifuge the 15 mL tube in a swinging bucket rotor for 10 minutes at 750g at 4C.
SECTION: Cell Fixation
56. Remove and discard the supernatant. Fully resuspend each pellet in 100 μL of cold Cell Buffer with a P200 set to 100 μL and return to ice. 
SECTION: Cell Fixation
60. Add DMSO: 0.5 ul into 25 ul samples and gently flick tubes to mix. One minute later, add another 0.5 ul and flick to mix, then after another minute add a final 0.5 ul for a total volume of 1.5 ul. Mix by gently pipetting 5x. Avoid creating bubbles.
SECTION: Cell Fixation
58. Count cells. Use 1:2 dilution factor, 10 ul + 10 ul dye.
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61. Place tubes in a Mr. Frosty for storage at -80C. The next day, move tubes to boxes in -80C racks.
SECTION: Cell Fixation
57. Take a 10 ul aliquot to dilute 1:2 with prepared 10 ul dye to manually count with a disposable hemacytometer and record numbers.
SECTION: Cell Fixation
59. Re-concentrate: spin cells 750g for 5 minutes and carefully take off supernatant until 15-25 ul are remaining. Resuspend (hopefully visible) pellet in the remaining ul and measure exact volume.