Agilent Seahorse XF Hu T Quick Start Guide

Quick Start Guide

Agilent Seahorse XF Hu T Cell Activation Assay Kit

One day prior to the assay (Day 1)

1. Ensure that the XF Analyzer is powered on and equilibrated to 37 °C (for a minimum of 5 hours).

2. Hydrate a sensor cartridge in sterile water in a 37 °C non-CO2 incubator overnight.

3. Prewarm a PDL-coated cell culture microplate in a 37 °C non-CO2 and non-humidified incubator overnight (for a minimum of 5 hours)

4. Design the experiment and create assay template in Wave

Day of assay (Day 2)

1. Complete sensor cartridge hydration by replacing water with XF calibrant (200 µL/well); incubate at 37 °C non-CO2 incubator for 1 hour.

2. Prepare 100 mL assay medium with XF supplements (Table 1). Warm up to 37 °C.

3. Centrifuge T cells and resuspend in the assay medium to the appropriate concentration (Table 2), and seed on XFe96 or XFp PDLcoated cell culture microplate.

4. Centrifuge the plate gently (e.g. 200x g for 1 min) to allow cells to adhere to the bottom of wells.

5. Gently add 130 µL assay medium to each well.

6. Incubate the plate in a 37 °C non-CO2 incubator for 45–60 minutes prior to the assay.

7. Prepare 10x working solution of the human CD3/CD28 activator, 2-deoxy-D-glucose (2-DG), and test compound (if desired) according to Table 3.

8. Load the 10x working solutions to injection ports following Table 4.

9. Open Wave and the pre-designed assay template. Click Start Run when you are ready.

10. Following calibration, Wave will display Load Cell Plate. Click Open Tray, then replace the Utility Plate with the Cell Plate.

11. Ensure that the lid is removed from the Cell Plate, then click

Load Cell Plate to start the assay.

Assay Media Component Volume (mL)

Seahorse XF RPMI Medium, pH 7.4

XF Glucose (1 M) 1.0 10 mM

XF Pyruvate (100 mM) 1.0 1 mM

XF Glutamine (200 mM) 1.0 2 mM

Table 1. Assay medium for XF Hu T Cell Activation Assay

Cell

T Cell Type

Naïve 4.0 x 10

Activated 2.0 x 10

Table 2. Recommended cell seeding densities for XF Hu T Cell Activation Assay

Reagent 10x Working Solution

Human CD3/CD28 Activator

2-DG Resuspend in 3 mL assay medium

Test compound 10x concentration in assay medium

Table 3. Preparation of 10x working solutions

Port

B 2-DG 22

C - - 2-DG 25

Suspension

(cells/mL)

Standard Assay Modulation Assay

10x Working

solution

CD3/CD28

Activator

Volume

(µL)

20 Test Compound 20

97 -

Seeding

Volume

(µL/well)

50 2.0 x 10

50 1.0 x 10

1:1 mix with assay medium

10x Working

CD3/CD28

solution

Activator

Final

Concentration

Final Cell

Density

(cells/well)

Volume

(µL)

Table 4. Volumes of 10x working solutions for ports.

Agilent Seahorse XF Hu T Cell Activation Assay Profiles

Standard assay Modulation assay

Activator or control

200 200

Baseline Baseline ModulationActivation Activation

150 150

100 100

PER (pmol/min)

50 50

0 0

25 2550 50

Time (minutes) Time (minutes)

2-DG 2-DG

Control

Activator

75 75100 100 125 150

Modulator or vehicle

PER (pmol/min)

CD3/CD28 activator

Ordering information

Part Number Product Description Compatible Analyzer

103759-100 Seahorse XF Hu T Cell Activation Assay Kit*, 200 tests All analyzers

Seahorse XF Hu T Cell Activation Assay 96-Well Pack, 200 tests

103766-100

Related Products

103576-100 Seahorse XF RPMI Medium, pH 7.4, 500 mL** All analyzers

103577-100 Seahorse XF 1.0 M Glucose Solution, 50 mL All analyzers

103578-100 Seahorse XF 100 mM Pyruvate Solution, 50 mL All analyzers

103579-100 Seahorse XF 200 mM Glutamine Solution, 50 mL All analyzers

103729-100 Seahorse XFe96 FluxPak Mini (PDL Plates), 6 assays XFe96/XF96 analyzers

103730-100 Seahorse XFe96 PDL Cell Culture Plates, 6 assays XFe96/XF96 analyzers

103721-100 Seahorse XFp FluxPak (PDL Plates) XF HS Mini, XFp analyzers

103722-100 Seahorse XFp PDL Cell Culture Miniplates XF HS Mini, XFp analyzers

103724-100 Seahorse HS Mini fluxPak (PDL Plates) XF HS Mini analyzer

103727-100 Seahorse HS PDL Miniplates XF HS Mini analyzer

(contains 1x Seahorse XF Hu T Cell Activation Assay Kit, 2x XFe96 FluxPaks with PDL microplates, 1x 100 mL Calibrant)

XFe96/XF96 analyzers

Control Vehicle Modulator 1 Modulator 2

* This product does not include any FluxPaks necessary for performing T cell activation assay. Relevant XF FluxPaks can be purchased separately. ** This medium can also be purchased together with the supplements/substrates listed in this table as bundled products (part number 103681-100).

Additional information

Agilent Seahorse XF Hu T Cell Activation Assay Kit User Guide:

https://www.agilent.com/cs/library/usermanuals/public/usermanual-agilent-seahorse-xf-human-tcellactivation-assay-kit-cell-analysis-5994-1811en-agilent.pdf

Agilent XF Learning Center:

www.agilent.com/en/products/cell-analysis/how-to-run-an-assay

Technical Support:

cellanalysis.support@agilent.com

For Research Use Only. Not for use in diagnostic procedures.

DE.274224537

This information is subject to change without notice.