Jun 09, 2026

363 KTEPH Study Worksheet 09: CAR T PE titrations: CAR T FITC titrations

This  protocol  is a draft, published without a DOI.
  • Terry Wightman1,2,3,
  • Terry Wightman4
  • 1University of Rochester Medical Center;
  • 2Flow Cytometry Shared Resource Lab;
  • 3Center for Advanced Research Technology;
  • 4University of Rochester
Terry Wightman
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Protocol Citation: Terry Wightman, Terry Wightman 2026. 363 KTEPH Study Worksheet 09: CAR T PE titrations: CAR T FITC titrations. protocols.io https://dx.doi.org/
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: June 09, 2026
Last Modified: June 09, 2026
Protocol  Integer ID: 318778
Keywords: regular titration, kteph study worksheet
Abstract
CAR T requires more work to be done than a regular titration. This is the protocol for that.
Guidelines
6. Aspirate supernatant, leaving 400uL.
7. Add 600uL to make final volume 1mL
8. Add 166uL’s to the 5 tubes labeled above, and bring up to 1mL
9. Put the remaining CAR T cells on ice. You may need this later
10. Spin 6 minutes @600xg @ RT with brake.
11. Label 6 tubes for SS comp controls
a. Unstained beads
b. CD3 BV510
c. CD45 AF647
d. X2 CAR T PE or CAR T FITC (which ever you’re titrating)
e. DAPI
12. When spin is done aspirate as close to pellet without disturbing it
13. Add 5uL of the trustain block
14. Incubate on ice 10 minutes
15. Add 1 drop of OneComp beads to the 3 SS comp controls
a. Add 49.375uL of HSM and .625uL of the CD45
b. Add 45 of HSM and 5uL of CD3
16. Add 100uL of the TC made for the first part above to all cells
17. Mix well
18. Incubate on ice 10 minutes
19. Do the following 2 dilutions:
a. Start with 1uL neat into 149uL HSM
b. Do a 1:10 dilution and use 1uL of this into 149uL HSM
20. Incubate on ice for 10 more minutes
21. Add up to 1mL of HSM to each
22. Spin 6 minutes @600xg @4 with brake.
23. Aspirate and transfer to a 5mL round bottom tube
24. Add 1uL of DAPI to the appropriate tubes ~5 minutes prior to running
25. Run on Janice

Volume of WS titration:
26. Thaw 1 vial of RC#3 by taking it out of the LN2 tank and swirling it around in a 37°C water bath until there are a few crystals left in vial
27. Add the total contents of vial into 10mL’s of HSM, ensure you rinse the vial with 1mL HSM out after taking the contents out
28. Spin 6 minutes @600xg @ RT with brake.
29. Label 3 tubes
a. 2uL
b. 1uL
c. .5uL
30. Aspirate until 200uL is left (take out 10.8mL)
31. Add 66uL to each of the 3 tubes labeled in step 30
32. Fill 15mL tube up with 3 mL’s and add 1mL to each of the 3 tubes
33. Spin 6 minutes @600xg 4°C with brake.
34. Aspirate as close to pellet without disturbing it
35. Add 5uL of truestain to each of the samples
36. Incubate 10 minutes on ice
37. Add the appropriate amount of FITC or PE WS to each tube
a. 2uL
b. 1uL
c. .5uL
38. Add 100uL of the TC made in the first section to each
39. Incubate both 20 minutes on ice
40. Add up to 1mL of HSM to each
41. Spin 6 minutes @600xg @ RT with brake.
42. Aspirate and transfer to a 5mL round bottom tube
43. Add 1uL of DAPI to the appropriate tubes ~5 minutes prior to running
44. Run on Janice using the same experiment used earlier.
Materials
Reagent/Supplies:
- HSM
- CAR T PE (BD/624255)
- CAR T FITC (BD/624201)
- CD3 BV510 (BD/564713, Lot # 5112025)
- CD45 AF647 (BD/557833, Lot # 4053477)
- DAPI (Invitrogen/D1306)
Reagent Preparation
Working solution titration: Note – this is for both PE and FITC
- 1:10 WS:
- Add 18uL of HSM into a 1.5mL tube
- Add 2uL of CAR T PE into the 18mL’s
- 1:5 WS: use the 1:5 WS already made, but if you need to make see below
- 16uL of HSM into a 1.5mL tube
- Add 4uL of CAR T PE
- 1:2.5 WS:
- Add 12uL of HSM into a 1.5mL tube
- Add 8uL of CAR T PE
- TC for both sections
- CD45 = 5
- CD3 = 40
- HSM = 755
Volume of WS titration
Note – this is for both PE and FITC
- Label the following 3 tubes
- 2uL
- 1uL
- .5uL
Procedure
Working solution titration:
Clean Hood.
Retrieve the CAR T cell above and thaw with 37°C water bath.
Transfer thawed samples to 15mL tubes containing 10mL HSM, rinsing original tubes/caps with 1mL of HSM.
Spin 6 minutes @600xg @ RT with brake.
Label 4 tubes
a. no WS
b. 1:2.5 WS
c. 1:5 WS
d. 1:10 WS
e. Unstained cells
Aspirate supernatant, leaving 400uL.
Add 600uL to make final volume 1mL.
Add 166uL’s to the 5 tubes labeled above, and bring up to 1mL.
Put the remaining CAR T cells on ice. You may need this later.
Spin 6 minutes @600xg @ RT with brake.
Label 6 tubes for SS comp controls
a. Unstained beads
b. CD3 BV510
c. CD45 AF647
d. X2 CAR T PE or CAR T FITC (which ever you’re titrating)
e. DAPI
When spin is done aspirate as close to pellet without disturbing it.
Add 5uL of the trustain block.
Incubate on ice 10 minutes.
Add 1 drop of OneComp beads to the 3 SS comp controls
a. Add 49.375uL of HSM and .625uL of the CD45
b. Add 45 of HSM and 5uL of CD3
c. Add 50 HSM to unstained beads
Add 100uL of the TC made for the first part above to all cells.
Mix well.
Incubate on ice 10 minutes.
Do the following 2 dilutions for FITC/PE SS:
a. Start with 1uL neat into 149uL HSM
b. Do a 1:10 dilution and use 1uL of this into 149uL HSM
Add 1 drop of ProG beads to both SS dilutions
Label both SS
a. Neat FITC/PE
b. 1:10 FITC/PE
Incubate on ice for 10 more minutes. Which will be a total of 20 minutes for the previous steps
Add up to 1mL of HSM to each.
Spin 6 minutes @600xg @4 with brake.
Aspirate and transfer to a 5mL round bottom tube.
Add 1uL of DAPI to the appropriate tubes ~5 minutes prior to running.
Run on Janice. Using whatever SS looks the best for the FITC/PE
How much to use for WS:
Thaw 1 vial of RC#3 by taking it out of the LN2 tank and swirling it around in a 37°C water bath until there are a few crystals left in vial.
Add the total contents of vial into 10mL’s of HSM, ensure you rinse the vial with 1mL HSM out after taking the contents out.
Spin 6 minutes @600xg @ RT with brake.
Label 3 tubes
a. 2uL
b. 1uL
c. .5uL
Aspirate until 200uL is left (take out 10.8mL).
Add 66uL to each of the 3 tubes labeled in step 30.
Fill 15mL tube up with 3 mL’s and add 1mL to each of the 3 tubes.
Spin 6 minutes @600xg 4°C with brake.
Aspirate as close to pellet without disturbing it.
Add 5uL of truestain to each of the samples.
Incubate 10 minutes on ice.
Add the appropriate amount of FITC or PE WS to each tube
a. 2uL
b. 1uL
c. .5uL
Add 100uL of the TC made in the first section to each.
Incubate both 20 minutes on ice.
Add up to 1mL of HSM to each.
Spin 6 minutes @600xg @ RT with brake.
Aspirate and transfer to a 5mL round bottom tube.
Add 1uL of DAPI to the appropriate tubes ~5 minutes prior to running.
Run on Janice using the same experiment used earlier.