Intracellular staining for flow cytometry with suspension cells on the Guava EasyCyte or BD FACScan/LSRII/Aria

Required reagents: PBS, PBS+1%BSA (PBS+), 0.3%saponin in PBS+1%BSA (saponin-PBS+), 10% neutral buffered formalin, primary and secondary antibodies.

∑ Wash off media and resuspend cells in cold PBS+0.5% FBS or PBS+1%BSA (PBS+). Cell density should be ~250,000-500,000 cells/ml count using a haemocytometer.
∑ Wash 2x with PBS+ at 3.2 krpm (1000 g) for 5 mins. Aspirate out supernatant, leaving ~50 ul around cell pellet. Gently tap and resuspend cell pellet. This is the standard wash procedure.
∑ Add 250 ul of 10% neutral buffered formalin, mix and incubate cold for ~20 mins.
∑ Wash 2x with PBS+ and resuspend pellet in 0.3%saponin, freshly prepared in PBS+ (saponin-PBS+). Incubate cold for ~30 mins.
∑ Wash and resuspend cell pellet. Add titred primary Ab in a volume of 50 ul saponin-PBS+ (total volume ~100 ul). Concentrations vary across Ab/Ag pairs most commercial Abs can be used at a range of 1:250-1:1000. Incubate cold for 60-120 mins.
∑ Wash 3x with saponin-PBS+
∑ Resuspend cell pellet in 50 ul. Add secondary Ab (depending on Ab/flurophore usually 1:2500-1:5000) in 50 ul volume in saponin-PBS+.
∑ Incubate cold for ~60-90 mins.
∑ Wash 2x with saponin-PBS+ and 1x with PBS+. Resuspend in ~300 ul. Ensure that no clumps are present.
∑ Acquire samples on the Guava EasyCyte using the ExpressPlus module.
Additional cell numbers may be required for BD machines.

For this intracellular staining assay for using the Guava easycyte did you use any of the express reagents that Guava offered (like the 7-AAD or the goat antibody)? Would it be possible to test whether a population that is supposed to be clonal is actually clonal or not using this intracellular staining? The cell population is supposed to be expressing IgG and I want to test whether there is more than one cell population in the colony. Thanks!

How does fixing and permeabilizing with 10% buffered formalin and saponin compare to the other methods? What are the advantages of using these materials versus using methanol-acetone, paraformadehyde-Triton, paraformadehyde-Methanol, and PEM-ethanol? I will be using suspension CHO, HEK293, and PerC6 cells for the intracellular staining. Please let me know. Thanks!

jp4 said:

How does fixing and permeabilizing with 10% buffered formalin and saponin compare to the other methods? What are the advantages of using these materials versus using methanol-acetone, paraformadehyde-Triton, paraformadehyde-Methanol, and PEM-ethanol? I will be using suspension CHO, HEK293, and PerC6 cells for the intracellular staining. Please let me know. Thanks!

Ideally you should check out multiple fixation methods, but I can probably help you for two of the cell lines:
If I've got your expt protocol right, you want to screen for Ab production by CHO or HEK293 by intracellular staining?
The 10%NBF (or 4%PFA)+0.3% saponin works fine for both these cell types.
Triton can have fluorescence using the standard 488 nm Argon laser, and its also harsher - hence saponin (you can also use Triton if you expect a srong signal, which should be the case for you)
Using ethanol or methanol for fixation is slow and painful - ice cold 80% EtOH needs to be slowly added drop by drop with continuous gentle vortexing - I prefer to avoid it whenever possible.
Also, I'd recommend you use BFA or monensin for 4/6 h prior to staining to minimize Ab secretion (I'm assuming you have a secretory tag in the vector, and are using these cells as Ab factories).

Hi! I've given you a general protocol to identify your Ig producing CHO cells - since they do not normally produce Ig, a specific Ab staining in viable cells would indicate production f the recombinant protein. Note that the Guava can only give you info about extent of Ig production and population based efficiencies. It will not enable you to isolate a clonal population, but can help you confirm that a population is clonal.

This protocol is not based on the one at the conference (with regards to the message I received).
Hope it helps.