Immunophenotyping of stem cell-like memory T (Tscm) cells in CAR T cell products using flow cytometry

MBTP 45 = Miltenyi Biotec-tested panel 45

This application protocol describes the analysis of stem cell-like memory T (Tscm) cell frequencies in chimeric antigen receptor (CAR) T cell products. To demonstrate the state-of-the-art panel, CAR T cells were generated within 12 days using the CliniMACS Prodigy® T Cell Transduction process and stained for Tscm cells. The data was analyzed using Miltenyi Biotec’s automated data acquisition and analysis Express Mode program “CAR T Cell Express Mode Package”.

Print protocol

Protocol

Staining protocol

Surface staining

Volumes given below are for up to 10⁶ nucleated cells. When working with fewer than 10⁶ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁶ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

Cell number was determined.
Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
A staining cocktail with all antibodies was prepared according to the dilutions mentioned in the respective data sheets.
100 µL staining cocktail was added to the cells.
Cells were resuspended and incubated for 10 minutes in the dark at room temperature.
Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
Cells were resuspended in 500 µL of PEB buffer. 5 µL of 7-AAD Staining Solution was added and incubated for 5 minutes in the dark at room temperature right before flow cytometric analysis using the MACSQuant® Analyzer 10.

Tested antibody panel

Antibody panel for Tscm cell analysis

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD*	-	Viability	-	655-730 (blue)
CD3*	REA613	T cells	FITC	525/50 (blue)
CD4*	REA623	Helper T cells	VioGreen™	525/50 (violet)
CD8*	REA734	Cytotoxic T cells	APC-Vio® 770	750 LP (red)
CD45RO	REA611	T cell differentiation	APC	655-730 (red)
CD197 (CCR7)*	REA546	T cell differentiation	VioBlue®	450/50 (violet)
CD95 (FAS)	REA738	T cell differentiation	PE	585/40 (blue)
CD62L*	145/15	T cell differentiation	PE-Vio 770	750 LP (blue)

* Alternative CE-IVD/ASR reagents available.

Gating strategy

Gating strategy showing the analysis of stem cell-like memory T (Tscm) cell frequencies in CAR T cell products. Lymphocytes were identified by a SSC^low gate which also excluded debris (A). Dead cells were excluded by gating on 7-AAD⁻ cells (B). CD3⁺ cells were identified (C) and discriminated in CD4⁺CD8⁻ and CD4⁻CD8⁺ cells (D). CD4⁺ and CD8⁺ T cells were further characterized by expression of CD197 and absence of CD45RO. CD4⁺ T cells are shown here as an example (E). CD45RO⁻CD197⁺ cells were then discriminated in CD95⁻ naive T cells (Tn) and CD95⁺ Tscm cells (F).

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Matching products:

130-111-568

7-AAD Staining Solution

1 mL

USD 79.00

130-110-681

CD8 Antibody, anti-human, APC-Vio
^®
770
, REAfinity™

REA734

100

tests

in 200 µL (1)

USD 340.00

130-113-230

CD4 Antibody, anti-human, VioGreen™, REAfinity™

REA623

100

tests

in 200 µL (1)

USD 361.00

130-113-621

CD62L Antibody, anti-human, PE-Vio
^®
770

145/15

100

tests

in 200 µL (1)

USD 345.00

130-113-004

CD95 (FAS) Antibody, anti-human, PE, REAfinity™

REA738

FC, MICS, IF, IHC

100

tests

in 200 µL (1)

USD 297.00

130-113-138

CD3 Antibody, anti-human, FITC, REAfinity™

REA613

FC, MICS, IF, IHC

100

tests

in 200 µL (1)

USD 255.00

130-113-556

CD45RO Antibody, anti-human, APC, REAfinity™

REA611

FC, MICS, IF, IHC

100

tests

in 200 µL (1)

USD 308.00

130-117-353

CD197 (CCR7) Antibody, anti-human, VioBlue
^®
, REAfinity™

REA546

100

tests

in 200 µL (1)

USD 340.00

Materials

Reagent and instrument requirements

Antibody panel for Tscm cell analysis

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD*	-	Viability	-	655-730 (blue)
CD3*	REA613	T cells	FITC	525/50 (blue)
CD4*	REA623	Helper T cells	VioGreen™	525/50 (violet)
CD8*	REA734	Cytotoxic T cells	APC-Vio® 770	750 LP (red)
CD45RO	REA611	T cell differentiation	APC	655-730 (red)
CD197 (CCR7)*	REA546	T cell differentiation	VioBlue®	450/50 (violet)
CD95 (FAS)	REA738	T cell differentiation	PE	585/40 (blue)
CD62L*	145/15	T cell differentiation	PE-Vio 770	750 LP (blue)

* Alternative CE-IVD/ASR reagents available.

Additional reagents used

PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA
Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
(Optional) MACSQuant Calibration Beads (# 130-093-607)
(Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)

Trademarks and disclaimers

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

If not otherwise specified, all marks displayed on the Miltenyi Biotec Internet site and documents are subject to the trademark rights of Miltenyi Biotec, including each of Miltenyi Biotec's primary brands, its model name plates, and its corporate logos and emblems.

For copyright note and a complete list of trademarks visit www.miltenyibiotec.com/legalnotes.

Matching products:

130-110-681

CD8 Antibody, anti-human, APC-Vio
^®
770
, REAfinity™

REA734

100

tests

in 200 µL (1)

USD 340.00

130-111-568

7-AAD Staining Solution

1 mL

USD 79.00

130-113-004

CD95 (FAS) Antibody, anti-human, PE, REAfinity™

REA738

FC, MICS, IF, IHC

100

tests

in 200 µL (1)

USD 297.00

130-113-230

CD4 Antibody, anti-human, VioGreen™, REAfinity™

REA623

100

tests

in 200 µL (1)

USD 361.00

130-113-138

CD3 Antibody, anti-human, FITC, REAfinity™

REA613

FC, MICS, IF, IHC

100

tests

in 200 µL (1)

USD 255.00

130-113-621

CD62L Antibody, anti-human, PE-Vio
^®
770

145/15

100

tests

in 200 µL (1)

USD 345.00

130-113-556

CD45RO Antibody, anti-human, APC, REAfinity™

REA611

FC, MICS, IF, IHC

100

tests

in 200 µL (1)

USD 308.00

130-117-353

CD197 (CCR7) Antibody, anti-human, VioBlue
^®
, REAfinity™

REA546

100

tests

in 200 µL (1)

USD 340.00

Immunophenotyping of stem cell-like memory T (Tscm) cells in CAR T cell products using flow cytometry

MBTP 45 = Miltenyi Biotec-tested panel 45

Protocol

Materials

Protocol

Staining protocol

Surface staining

Tested antibody panel

Antibody panel for Tscm cell analysis

Gating strategy

7-AAD Staining Solution

CD8 Antibody, anti-human, APC-Vio
^®
770
, REAfinity™

CD4 Antibody, anti-human, VioGreen™, REAfinity™

CD62L Antibody, anti-human, PE-Vio
^®
770

CD95 (FAS) Antibody, anti-human, PE, REAfinity™

CD3 Antibody, anti-human, FITC, REAfinity™

CD45RO Antibody, anti-human, APC, REAfinity™

CD197 (CCR7) Antibody, anti-human, VioBlue
^®
, REAfinity™

Materials

Reagent and instrument requirements

Antibody panel for Tscm cell analysis

Additional reagents used

Trademarks and disclaimers

CD8 Antibody, anti-human, APC-Vio
^®
770
, REAfinity™

7-AAD Staining Solution

CD95 (FAS) Antibody, anti-human, PE, REAfinity™

CD4 Antibody, anti-human, VioGreen™, REAfinity™

CD3 Antibody, anti-human, FITC, REAfinity™

CD62L Antibody, anti-human, PE-Vio
^®
770

CD45RO Antibody, anti-human, APC, REAfinity™

CD197 (CCR7) Antibody, anti-human, VioBlue
^®
, REAfinity™

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