I would like to do intracellular staining of spleenocytes. I need to prepare the reagents for fixation and permeabilization reagents. I also need the detailed staining protocol for flow.
Perm buffer can be variable, as different batches of saponin should be tested. But you can make it and store it at -20. Fixation buffer is just 2-4% Paraformaldehyde in PBS, and should be made fresh 1x/month as it degrades.
Permeability Buffer is
PBS
1%Fetal Calf SErum
0.1% Sodium Azide (TOXIC and bad combination with lead pipes)
0.1% Saponin
Filter (0.2um)
pH to 7.4-7.6 (est. 2 drops NaOH)
Fix: The trick is to warm the PBS (e.g. 60C waterbath, or heat/stir on very low 1-2hrs, or O/N), but this should be done in a hood, as formaldehyde fumes are noxious.
Wash your cells three time in Perm BUffer after 20-30min fix, Stain in Perm buffer. Then, wash 3 more times in Perm Buffer.
Good luck!!
We use BD Bioscience Cytofix/Cytoperm Fixation/Permeabilazation kit (#554714) that uses formaldehyde for fixation and saponin for permeabilization. I'm sorry, but I don't know their recipes. I've attached the protocol that we have used successfully for intracellular staining for flow that was adapted from BD Bioscience's protocol that I have also attached.
Hi, according to my experience "BD Bioscience Cytofix/Cytoperm Fixation/Permeabilazation kit" is milder than "Fixation/Permeabilization Concentrate and Diluent" from eBioscience. You need longer permeabilization time if you use BD (overnight maybe) but for eBioscience, the sample will be permeabilized within 1h (40min). But you could see on FACS that eBioscience's kit will make cells shrink (smaller)
Thanks. Yes, I've previously used the commercially available kits as you mentioned, but now I would like to use the in-house-made reagents for fixation and perm. for intracellular staining to be assessed by flow. I would appreciate if you have the recipes of the fixation and perm reagents and the experimental protocol for intracellular staining with those reagents in murine spleenocytes. Thanks again.
Perm buffer can be variable, as different batches of saponin should be tested. But you can make it and store it at -20. Fixation buffer is just 2-4% Paraformaldehyde in PBS, and should be made fresh 1x/month as it degrades.
Permeability Buffer is
PBS
1%Fetal Calf SErum
0.1% Sodium Azide (TOXIC and bad combination with lead pipes)
0.1% Saponin
Filter (0.2um)
pH to 7.4-7.6 (est. 2 drops NaOH)
Fix: The trick is to warm the PBS (e.g. 60C waterbath, or heat/stir on very low 1-2hrs, or O/N), but this should be done in a hood, as formaldehyde fumes are noxious.
Wash your cells three time in Perm BUffer after 20-30min fix, Stain in Perm buffer. Then, wash 3 more times in Perm Buffer.
Good luck!!
thank you very much Robin and Dr. Bruce. Can you tell me the exact amount of each to make the solution?
Robins details are spot on though I have found that replacing the FCS with BSA V is an improvement on the original formulation. The composition of saponin from Quillaja bark is Extremely variable so can I recommend trying Sigma's Saponin S7900 which I find gives highly reproducible results
Thanks everyone for these helpful tips. But this certainly varies on what we are staining for right? Is staining for phospho-kinases which are expressed only transiently same as for cytokines for example where you can block their secretion, fix stain and get them? Can some one enlighten in the case of phospho-kinases?
Sir,, please find the protocol form ORIGINE.
If it works let me know,even if it is not working ,plz let me know.. so that i will make decision for my FC assay.
Wish you good luck ..
Robin's protocol is good for intracellular staining. If you buy the molecular grade of saponin, rather than quillaja bark, which is highly variable and can lead to fungal contamination, without the azide[0.025%] or filtering. However, this is not usually sufficient for the intranuclear staining you actually want. You can either extend your perm/staining period 18-24hours or change your surfactant - try 0.1% Triton X-100. I also tend to fix with ethanol for histone staining, but surface staining may be reduced, so you need to check. Here is the protocol I've used.
Thanks everyone. I have a quick question. I use BD cytofix (has 4.2% formaldehyde) 15min for fixation followed by 0.1% saponin (1hr RT) treatment for permeabilization. This protocol works fine for murine spleen T cells but did not work with intrahepatic myeloid cells. I really dont know what went wrong. Does saponin treatment influence autofluorescence and do i need to treat the single color compensation cells with saponin as well?
Thanks
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