DTT Processing

1. Weigh the sputum sample and transfer the whole sputum to a 15/50ml conical vial. Record the weight on the CASA sputum processing worksheet.
2. Prepare a solution of 10% of 1 molar DTT in 1xPBS (which is a .1 molar DTT concentration)
   1. A vial of DTT is good for 1 week after first use. Mark the vial with the date opened.
   2. A 10% DTT solution may be used up to 12 hours after preparation if kept at 4°C.
   3. Dilute the sputum sample 1:1 in 10% DTT. Invert to mix. (.05 molar DTT solution)
   4. Add DNAse (from stock 1mg/mL) 1:10 ratio.
   5. Agitate at 37°C/150rpm for 15 minutes, checking the sample at 5-minute intervals to ensure breakdown. Remove when the sample is no longer viscous.
      1. Check viscosity by slowly rotating the vial and observing adhesion of sputum to the sides.

Centrifugation/Resuspension

1. Centrifuge the DTT-processed sputum at 300xg for 5min.
2. Decant DTT supernatant.
3. Resuspend pellet in a volume of FACS Buffer equal to original sample volume          

                  a.    1% BSA

                  b. 0.001% EDTA

                  c. 1X PBS

1. Spin down at 300Xg for 5 min, and re-suspend in above (additional wash step)
2. 1.         1% BSA
3. 1.         0.001% EDTA
4. 1.         1x PBS
5. Place a BD Cell Strainer in the top of a 50ml conical labeled with subject ID and date. Using a serological pipet, gently apply the sample to the strainer, using the pipet to aid flow through the mesh. For samples over 7g, use two strainers; switch to new filter about halfway through the sample.
6. Keep strained sample on ice while doing cell count.

Cell Count

1. Mix 20ul of sample with 20μl Turks Solution*.
   1. Store the rest of the sputum sample at 4°C while performing cell count.

*10mg Crystal Violet

3mL Glacial Acetic Acid

to 100ml with ddH2O

Protocol for CyTOF stains:

1. Centrifuge cells of interest (~3 million) for 5 minutes and resuspend in PBS (1 million cells per mL).
2. Add cisplatin at a concentration of 25 uM (1:10,000 of the 250mM stock). 1ul cisplatin in 10mL CyPBS.  Resuspend the cells in 100ul of this mixture for incubation.
3. Incubate for EXACTLY 1 minute at RT
4. Quench reaction with media with 10% FBS
5. Wash approximately 3 million cells in 600ul of CyFACS buffer for 10 minutes at RT.
6. Add 50ul of the primary CyTOF antibody cocktail to each tube and incubate for 30 minutes.
7. Wash twice with 500uL of CyFACS buffer for 10 minutes at RT.
8. Add 50ul of the secondary CyTOF antibody cocktail to each tube and incubate for 30 minutes.
9. Wash twice with 500uL of CyFACS buffer for 10 minutes at RT

10. Add 100ul of 2% PFA and keep cells at 4°C O/N (or up to 2/3 days maximum)

11. Wash cells with 500ul CyFACS buffer and continue with protocol, or keep in CyFACS buffer in the fridge.

12. If continuing with protocol: wash with 500ul CyFACS buffer at 4°C for 10 minutes.

13. Add 100ul of 1x saponin perm buffer (9mL PBS + 1mL BSA + 100 ul NaAz + 0.05g saponin) for 45 minutes on ice.

14. Wash with CyFACS for 10 minutes at RT then 10 minutes at 4°C.

15. Stain with Intercalation buffer (1mL Intercalation buffer + 1:5000 dilution of Iridium Nucleic Acid) for 20 minutes at RT.

16. Wash 10 minutes at RT with 500ul CyFACS (keep in CyFACS buffer while transporting to HIMC)

17. Repeat wash twice with MilliQ water before running on CyTOF

Antibody mix

• Blocking antibody (mouse or rat serum)

Included in kit:

• 170Er- CD3 (1:100)
• 145Nd- CD4 (1:200)
• 146Nd- CD8 (1:200)
• 159Tb- CD11c (1:100)
• 160Gd- CD14 (1:100)
• 148Nd- CD16 (1:100)
• 142Nd- CD19 (1:100)
• 147Sm- CD20 (1:100)
• 167Er- CD27 (1:100)
• 154Sm- CD45 (1:100)
• 169Tm- CD45RA (1:100)
• 151Eu- CD123 (1:100)
• 174Yb- HLA-DR (1:100)

Not included in kit:

• 141Pr- CCR6 (1:100)
• 176Yb- CD127 (1:100)
• 172Yb- CCR4 (1:100)
• 171Yb- CD1c (1:100)
• 168Er- FcER1 (1:100)
• 164Dy- CD161 (1:100)
• 156Gd- CXCR3 (1:200)
• 144Nd- CD11b (1:100)
• 149Sm- CD25
• 165Ho- CD45RO
• 166Er- CRTH2
• 172Yb- CCR3
• 175Lu- ST2
• 143Nd- CD117 (ckit)
• 150Nd- Biotin
• 152Sm- CD66b