Ramos B cell calcium flux was measured as described (75 (link)). Protein tetramers were formed at a 4:1 molar ratio of protein to streptavidin (Invitrogen). Ramos cell lines stably expressing DH270 UCA, DH270.1, or CH65 IgM (76 (link)) were passaged (1:10) 4 days before calcium flux experiments. On the day of the experiment, cells with >95% viability were resuspended at 106 cells ml−1 in 2:1 ratio of RPMI media (GIBCO) + FLIPR Calcium 6 dye (Molecular Devices). Cells were plated in a U-bottom 96-well tissue culture plate (Costar) and incubated at 37°C 5% CO2 for 2 hours. In a black clear-bottom 96-well plate (Costar) containing 50 μl of RPMI media (GIBCO) + FLIPR Calcium 6 dye (Molecular Devices) (2:1 ratio) either 0.1 nmol of proteins or 50 μg ml−1 of anti-human IgM F(ab’)2 (Jackson Immuno) were added (based on a 100-μl volume). Using a FlexStation 3 multimode microplate reader (Molecular Devices), 50 μl of supernatant containing cells were transferred into the 50 μl of media containing protein or anti-human IgM F(ab’)2 (Jackson Immuno) and continuously read for 5 min. Relative fluorescent value units were background-subtracted and the data expressed as percentage of the IgM maximum signal (% IgM max).
Flipr calcium 6 dye
Manufactured by Molecular Devices
The FLIPR Calcium 6 dye is a fluorescent indicator used for the detection and measurement of intracellular calcium levels in live cells. It is designed to provide a sensitive and reliable method for monitoring calcium-mediated cellular responses.
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Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Black 96 well plate, by Thermo Fisher Scientific (1 mentions)
Rpmi media, by Thermo Fisher Scientific (1 mentions)
Anti human igm f ab 2, by Jackson ImmunoResearch (1 mentions)
Flexstation 3 multi mode microplate reader, by Molecular Devices (1 mentions)
Flexstation3 multimode plate reader, by Molecular Devices (1 mentions)
Probenecid, by Thermo Fisher Scientific (1 mentions)
Flipr tetra system, by Molecular Devices (1 mentions)
6 protocols using flipr calcium 6 dye
1
Ramos B cell calcium flux assay
2
Calcium Flux Assay for Thymic Fractions
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L1.2 stable lines were seeded at 300,000 cells/well in black clear-bottom 96-well plates and incubated with FLIPR Calcium 6 dye (Molecular Devices) for 2 hours at 37°C following the manufacturer’s recommendations. As mentioned above, thymic ApoBD, MVs, and SN fractions used in calcium flux assays were prepared in calcium assay buffer (1:1 vol:vol RPMI-Glutamax 10% FBS:HEPES (20 mM) in HBSS) and used without further dilution. Calcium flux signals were recorded for 180 seconds in a FlexStation 3 plate reader (Molecular Devices) using the instrument’s built-in pipettor to automatically dispense 25 μl of buffer, the appropriate recombinant chemokine agonists (50 nM in assay buffer), or thymic fractions at 20 seconds after the initiation of the recording.
3
Calcium Signaling Responses in Mast Cells
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LAD2 (0.2 × 105 cells in 100 μL) and RBL-2H3 (0.5 × 105 cells in 100 μL) cells were plated in 0.1% SIR-BSA. FLIPR® Calcium 6 dye (Molecular Devices) was reconstituted as per manufacturer's protocol, combined with cells (1:1 ratio) and incubated for 2 h at 37°C and 5% CO2. Cells were then stimulated with CST-14, LL-37, substance P, compound 48–80, or (R)-Zinc-3573 using the FlexStation® 3 Flex-protocol, and changes in fluorescence were measured over a 120 s period. For assays involving inhibitors, cells were incubated with SKF, YM, Nifedipine, or A425619 for 30 min prior to agonist stimulation. Phosphate-buffered saline (PBS) was used as the control vehicle used for all the inhibitors. Excitation and emission wavelengths were 485 and 525 nm, respectively. Fluorescence data were normalized to maximal response values.
4
Calcium Flux Assay for VRC01 bNAb-Expressing B Cells
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Ramos Burkitt’s lymphoma B cells stably expressing the BCR for the VRC01 bNAb were a gift from Daniel Lingwood (Massachusetts Institute of Technology, Boston, MA) (60 (link)). The line was cultured in RPMI 1640 medium (Thermo Fisher Scientific) containing 15% heat-inactivated fetal bovine serum (FBS; Gibco) and 1× penicillin-streptomycin and maintained at 37°C in 5% CO2. For experimental use, the cells were resuspended at 2 × 106 per ml in RPMI 1640 medium supplemented with 15% fetal bovine serum (FBS) and the FLIPR Calcium 6 dye at the manufacturer-recommended final concentration (Molecular Devices) and then incubated in a 96-well U-bottom tissue culture plate (Costar) for 2 h at 37°C in air containing 5% CO2. The B41 SOSIP.v4.1 soluble or IO-NP trimers (10 μg of Env content) in 50 μl of the above-described medium, with the Calcium 6 dye still present, per the manufacturer’s recommendation, were added to a black 96-well plate with a transparent base (Thermo Fisher Scientific). The dye-loaded B cells (50 μl) were then added to the same wells, and the fluorescence signal (excitation at 494 nm, emission at 516 nm) was immediately recorded every 5 s for 3 min using an EnSpire multimode plate reader.
5
Mast Cell Calcium Signaling Assay
6
Measuring CCR8-mediated Calcium Flux
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Human or mouse CCR8-CHO cells were seeded and incubated for 2 hours at 37 C with FLIPR Calcium 6 dye (Molecular Devices) and probenecid (Thermo Fisher Scientific). After incubation, antibodies were added at room temperature for 15 minutes. 10 nmol/L of recombinant human or mouse CCL1 (R&D Systems) was added to samples through a FLIPR Tetra system (Molecular Devices) for calcium flux fluorescent signal measurement.
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