Detection of phosphoepitopes by Flow cytometry

FACS phospho-staining: ex vivo staining of phosphoepitopes

• FACS buffer : PBS, 2% SVF, 0,09% azide
• Lyse/Fix ref#558049
• Perm III ref# 558050

Dilute lyse fix (1/5 in distilled water). Prewarm at 37°C.

Place PermIII at -20°C (contains 90% methanol) and FACS buffer on ice.

Cool the centrifuge.

Upon harvest, place the organ in ice cold medium and keep on ice. Process the organ as quickly as possible, working on ice.

After organ dissociation, stain around 3x10⁶ cells/staining.

Stain the cells for 10 min on ice in 100µl of the following mix :

Wash 1x with ice cold FACS buffer.

Resuspend the pellet in 100µl of 1x Lyse/fix prewarmed at 37°C. Incubate for 10 min at 37°C.

Wash 1x with ice cold FACS buffer.

Break the pellet by vortexing quickly. Add 150µl ice cold PermIII drop by drop. Resuspend using a pipette. Incubate for 30 min on ice.

Wash 3 times using ice cold FACS buffer to rehydrate the cells.

Stain the cells for 30-45 min at room temperature in 50µl of the following mix :

The volumes indicated in this protocol apply for stainings done in 96-well plates (conical bottom). Alternatively, stainings can be performed in 15ml Falcon tubes (this allows staining of larger quantities of cells) and volumes adapted accordingly.

FACS phospho-staining: Phosphoepitopes staining following short-term stimulation

• FACS buffer: PBS, 2% SVF, 0,09% azide
• Complete medium RPMI1640+Glutamax (Gibco 61870), 10% FCS, 1mM Pyruvate, 10mM HEPES, Penyl/Strep, 50µM 2-MercaptoEthanol
• Lyse/Fix BD ref#558049, can be replaced by 2% PFA
• Perm III BD ref# 558050, can be replaced by 90% Methanol / 10% H2O

Dilute Lyse/fix (1/5 in distilled water). Pre-warm at 37°C.

Place PermIII at -20°C (contains 90% methanol) and FACS buffer on ice.

Cool the centrifuge.

Following spleen harvest, prepare the cell suspension at RT and stain 2-3x10⁶ cells/staining for surface markers (10 min staining @RT in complete medium).

CD3 APCef780 clone 2C11

CD19 APCef780 clone 1D3

TCRb APCef780 clone H57

CD49b APC or CD49b BV421 clone DX5

Rmk: other markers like CD45s can be added to barcode several populations. I tested CD45.1 BV605, CD45.2 A700, CD45.2 BV510, CD45.2 BV786.

Wash in complete medium once and resuspend in 2-3x10⁶ cells/100µl of complete medium. Place in a 15ml Falcon and put in a water-bath at 37°C for 5-10min.

Do the NK1.1 stimulation as follow: add NK1.1 biot (5B6) at 5µg/ml final and wait for 1 min 30 sec. Then add Strepta (6F10) at 10µg/ml to cross-link the NK1.1. Start the kinetic from this point on.

To stop the stimulation, fix the cells by adding 10 volumes of 1x Lyse/fix pre-warmed at 37°C (i.e. 1ml if the stimulation is performed in 100µl). Incubate for 10 min at 37°C.

Wash 1x with ice cold FACS buffer.

Break the pellet by vortexing quickly. Add 1ml ice cold PermIII drop by drop. Resuspend carefully. Incubate for 30 min on ice.

Wash 3 times using ice-cold FACS buffer. Transfer the cells in a 96-well plate.

Stain the cells for 30-45 min at room temperature in 50µl of the desired anti-phospho Antibody mix.