Calcium flux detection:

- Indo-1-AM: 1mM = 1mg/ml (1000x, endconc. 1ug/ml)

(Mol. Probes/invitrogen, cat. # I1226)

- HEL: 10mg/ml stock in water (SIGMA, cat. # L6876)

- Anti-IgM: F(ab’)2 fragment goat anti-mouse IgM) 1.3 mg/ml

(Jackson Immuno cat # 115-006-075)

- CLM (cell loading medium): RPMI-1640, 5%FCS, pen/strep

• Isolate splenocytes from HEL Tg or use cell line (Bal17 or other)

• Spin down, do RBC lysis (in case of splenocytes)

• Wash cells and count

• take desired amount of cells and spin down

• Resuspend cells to 1 x 10⁷ /ml in CLM

• add indo-1/AM to endconc. of 1 ug/ml (=1uM) and incubate for 30 min at 37C in dark (shake a few times for even loading)

If you want to do a staining on the cells:

• Wash and spin down, resuspend again to 1x10⁷/ml

• stain for 15 min at RT in dark

• wash cells

• Dilute cells to 1x10⁶ cells per ml (you can use a bit more concentrated if desired but 1x10⁶ cells/ml is enough for a cell line)

• add 1ml per facs tube

• Analyze The ratio of indo-1 violet/blue using UV laser (I use Vantoo machine with the DIVA software)

• Collect data for 30-50 sec to establish baseline violet/blue ratios (bound ~420nm, free ~510nM)

• Stimulate cells with HEL Ag, or anti-IgM F(ab’)2 (or p.c. Ionomycin) and collect data for a total of 5-6 min.

• Range of HEL ag I use: effects on splenocytes of HEL tg start at +/- 10ng/ml but you can use much more depending on purpose (i.e 250ng/ml)

• Range of anti-IgM I use: starting at 0.5 ug/ml up to 5 ug/ml is optimal range for BAL17 cells

• You can use Ionomycin as a positive control (all cells should respond)

· Data is analyzed afterwards using flowjo software