Surface staining and calcium flux on mouse splenocytes.

Materials

• Cell Loading buffer (CLB), hanks w/o phenol red + 1% FBS
• Rat anti mouse CD8 PE (Invitrogen MCD0804)
• Hamster anti mouse CD3 (Invitrogen HM3400)
• Indo-1 AM ester Special packaging (Invitrogen I-1223)
• Ionomycin (Invitrogen I-24222)
• Pluronic F-127 20% w/v in DMSO (InvitrogenP3000MP)

Cell Preparation

• Mouse splenocytes were harvested and suspended at about 5x106 / ml in CLB. We suspended one mouse spleen in 20 ml of CLB, then put 10 ml of the cell suspension in each of the 15 ml conical tubes.
• The indo-1 was prepared by adding 50 µl of dry DMSO and 5 µl of Pluronic F-127 to the special packaging tube containing 50 µg of indo-1AM. The tube was vortexed for 5 seconds. Then 27.5 µl of this mixture was added to each of the 15 ml conical tubes containing the cells. This gave a final concentration of 2.5 µM indo-1.
• The cells were then incubated for 1 hour in a water bath at 37 °C, the tubes were inverted occasionally.
• The tubes were then centrifuged at 500 g for 10 minutes, all but 250 µl of the supernatant was removed and the cells were gently resuspended.
• The anti CD8 PE and anti CD4 FITC were added in both tubes 5 µl (0.5 µg). The tubes were incubated at room temperature for 10 minutes.
• Each tube had 10 ml of CLB added and was then centrifuged at 500 g for 10 minutes, the supernatant was aspirated and the cells in each tube were gently suspended in 1 ml of CLB. Cells were allowed to rest for 30 minutes.
• The samples were diluted 1:10 in CLB and aliquoted into 12X75 mm FACS tubes and kept at room temperature.

Flow Cytometry

• Prior to running on the instrument each sample was pre-warmed to 37 °C in a water bath for 5 minutes. Cells were then aspirated into the cytometer and the event rate adjusted to 2000 events per second. Acquisition was restarted and recorded for 10 seconds before samples were removed, the agonist added and the samples replaced on the instrument and acquisition continued for 5 minutes. The samples were kept at 37 °C by circulating warm water in a jacket surrounding the sample tube.
• The Ionomycin was diluted by dissolving 1 mg in 1ml dry DMSO. This was further diluted 1:10 in CLB before use and 10 µl was added to each sample making a final concentration of 2 µg/ml. The CD3 antibody was used in three doses, 0.4 µg/ml, 2 µg/ml and 4 µg/ml. This was achieved by diluting the stock antibody 1:10 with CLB for the lowest dose, 1:2 for the middle dose and using it neat for the highest dose. In all cases 10 µl was added. All samples were run in duplicate.
• Data were acquired using Becton Dickinson DIVA software and exported as FCS3.0 32 bit data files. The data were analysed using FlowJo v6, the median ratio of Indo-1-bound / Indo-1-unbound was plotted against time. The instrument used was a Becton Dickinson FACS Vantage with DIVA option. The Indo-1-bound signal was collected using a 390/22 bandpass filter and the Indo-1-unbound signal was collected using a 530/30 bandpass filter. The laser used to excite the Indo 1 was a Coherent Innova 90 Krypton tuned to the 350-356.4 nm UV lines. The primary laser was a Coherent Innova 90 Argon tuned to 488 nm.
• Please note. The sample line must be thoroughly cleaned after ionomycin by running DMSO for a few minutes otherwise the residue from the previous sample will contaminate the next sample.