Matt Spitzer, Iliana Tenvooren, Maggie Martins - 9.12.2016

CyTOF Staining Protocol

Staining Preparation

1.  Mix 2ml 2% saponin into 198ml PBS for 200ml 0.02% saponin stock.

2.  Distribute 900ul 0.02% saponin into deep well blocks in barcode plate layout (6*13 samples; 1*10 samples). Store at 4˚ along with remaining 0.02% saponin stock.

3.  Make master mixes for extracellular and intracellular staining cocktails.

4.  Make 150ml CSM supplemented with 1mM MgCl2 (30.5 mg of MgCl2•6H2O) and 15ml Benzonase.

Lysis

5.  Dilute 150ml of 1000x Thaw-Lyse concentrate in 150ml ddH2O to make 1x working stock (10ml per blood sample).

6.  If thawing blood samples, proceed below. Otherwise, skip to Step 7.

  1. Thaw blood samples in 4˚ fridge for 10 min.
  2. Remove and place in RT water bath for 15 min, but proceed to next step. Samples should be fully thawed.
  3. During the thaw step, arrange and label 50ml conical with cell strainer for blood. Add 5ml of 1x Thaw-Lyse buffer to each conical.
  4. Add blood samples through the strainer into respective FACS tube.
  5. Wash filter with 5ml 1x Thaw-Lyse. Gently vortex to mix.
  6. Incubate for 10 min at RT. Make 150ml of secondary lysis buffer from concentrate.
  7. Pellet cells at 600g for 5 min at RT. Aspirate off supernatant to 100ml.
  8. Add 10 ml secondary lysis buffer to each tube. Incubate 5 min at RT.
  9. Pellet cells at 600g for 5 min at RT. Aspirate off supernatant to 100ml. Pellet should now be nearly white.
  10. Resuspend pellet in 1ml CSM + 1mM Mg2+ + Benzonase (1:10,000) and count cells.

7.  Thaw other tissues in 4˚ fridge for 10 min.

8.  Remove and place in RT water bath for 15 min. Samples should be fully thawed.

9.  Pellet cells at 600g for 5 min at RT. Aspirate off supernatant to 100ml.

10.  Resuspend pellet in 1ml CSM + 1mM Mg2+ + Benzonase (1:10,000) and count cells.

11.  Transfer appropriate vol. for 1*106 cells into cluster tubes.

12.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml.

Barcoding

13.  Wash cells 1x PBS and pellet at 600g for 5 min at 4˚. Aspirate supernatant.

14.  Wash cells 1x 0.02% saponin in PBS and pellet at 600g for 5 min at 4˚. Aspirate supernatant.

15.  Add 100ul 0.02% saponin to each well using multichannel pipette.

16.  Thaw 1x barcode plate at RT using thaw block.

17.  Remove 0.02% saponin in deep well block from 4˚ fridge

18.  For each barcode plate, one row at a time, transfer 5 ml/well diluted BC reagents to 0.02% saponin in deep well block on ice. Pipet up/down. With p1000, pipet up/down 2x and immediately transfer 905ul into cluster tubes with cells. Pipet up/down 4x and gently vortex.

19.  Repeat for each row (13 total).

20.  Incubate RT for 15 min on shaker.

21.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

22.  Add 1ml CSM to each well.

23.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

24.  Add 1ml CSM to each well.

25.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

26.  Combine all samples for each series (13/10 samples) into 1 cluster tube. Wash cluster tubes with additional 300ul CSM and add to cluster tube.

27.  Pellet at 600g, 5 min, 4˚, and aspirate to residual volume of 100ml.

Extracellular Stain

28.  Add 1.99ml anti-CD16/32 (Pr141) to each tube and incubate RT for 5 min on shaker.

  1. For MMTV samples, add 1.2ml anti-CD16/32

29.  Add 184.77ml CSM to each tube.

  1. For MMTV samples, add 74.9ml CSM

30.  Add 38.24ml extracellular Ab cocktail to each cluster tube.

  1. For MMTV samples, also add 23.53ml antibody cocktail for final 200ml stain volume

31.  Incubate 30 min at RT on shaker.

32.  Add 1ml CSM to each tube.

33.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

34.  Add 1ml CSM to each tube.

35.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml. Vortex very well.

Permeabilization

36.  Add 1mL MeOH at 4˚ to each tube and incubate for 10 min at 4˚.

37.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

38.  Add 1ml CSM to each tube.

39.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

40.  Add 1ml CSM to each tube.

41.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

42.  Add 1ml CSM to each tube.

43.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

Intracellular Stain

44.  Add 217.18ml CSM to each tube.

  1. For MMTV samples, add 92.2ml CSM

45.  Add 7.82mL intracellular cocktail to each tube.

  1. For MMTV samples, add 4.81ml antibody cocktail as well as 3ml PyMT Ab

46.  Incubate 30 min at RT on shaker.

Intercalation

47.  Add 1ml CSM to each tube.

48.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

49.  Add 1ml CSM to each tube.

50.  Pellet at 600g for 5 min at 4˚. Aspirate supernatant to 100ml

51.  Make intercalator: 7ml PBS + 700ml 1.6% PFA and 1.4ml (1:5000) Ir in PBS.

52.  Add 1 mL intercalator to each tube.

53.  Incubate overnight at 4˚C.

Sample Acquisition

54.  Spin at 600g, 5 min at 4˚. Aspirate to 100ml.

55.  Add 1ml CSM. Spin at 600g, 5 min at 4˚. Aspirate to 100ml.

56.  Add 1ml ddH20. Spin at 600g, 5 min at 4˚. Aspirate to 100ml.

57.  Add 1ml ddH20. Spin at 600g, 5 min at 4˚. Aspirate to 100ml.

58.  Dilute CyTOF normalization beads (500x) in ddH2O, resuspend samples in bead solution, and run on CyTOF, aiming to collect 4*106 cells per sample