I am planning to detect few of B and T cell specific protein expression via flow cytomteric analysis. I've seen many has isolated PBMCs prior staining, however I know we can still stain and lyse RBCs and then detect the expression or gate them. So I wonder why we need to isolate PBMCs?
Oh! I'm a fan of whole blood staining...In my hands, however, better results were accomplished by first lysing (with NH4Cl solution, no formaldehyde) and then staining (provided a careful wash to eliminate the lysing solution). This is due to the fact that remaining leukocytes can be resuspended in the desired volume of staining buffer (I usually use D-PBS 3% FCS). Frequencies of cell subsets are the same between stain-than-lyse and lyse-than-stain techniques, but resolution (stain index) is higher in the latter case (using the same amount of antibodies). Thus, you can save liters of antibodies!
It is known that density-gradient isolation of mononuclear cells can affect relative frequencies or activate many cell subsets. In addition, maintaining the native cell composition (i.e. including neutrophils and eosinophils) can give you the possibility to easily determine the absolute cell number of the subset of interest by "dual-platform" assay. Before any cytometric determination, I lyse RBC of a small aliquot of blood with 2% acetic acid in H2O (20 microliters of blood by reverse-pipetting and 180 of diluted acetic acid) and count at the hemocytometric chamber. This tells the number of nucleated cells (i.e of CD45+ cells, unless erythroblasts/CD45- are present), while staining in parallel with other mixes one tube with CD45 gives the measure of how the leukocyte gate drawn in a physical parameters plot is close to that drawn in a SSC vs CD45 (i.e. the quality of lyse).
Obviously, each operator has to validate this protocol in his/her lab by the use of count-beads or true-count tubes. Personally, I have validated this protocol in a side-by-side comparison against the ADVIA hematology system present in my institute for EDTA vacutainers. Not only the absolute number of total leukocytes closely correlated with the manual count, but also the relative (and absolute) number of major leukocyte subsets including lymphocytes, monocytes, neutrophils and eosinophils.
Oh! I'm a fan of whole blood staining...In my hands, however, better results were accomplished by first lysing (with NH4Cl solution, no formaldehyde) and then staining (provided a careful wash to eliminate the lysing solution). This is due to the fact that remaining leukocytes can be resuspended in the desired volume of staining buffer (I usually use D-PBS 3% FCS). Frequencies of cell subsets are the same between stain-than-lyse and lyse-than-stain techniques, but resolution (stain index) is higher in the latter case (using the same amount of antibodies). Thus, you can save liters of antibodies!
It is known that density-gradient isolation of mononuclear cells can affect relative frequencies or activate many cell subsets. In addition, maintaining the native cell composition (i.e. including neutrophils and eosinophils) can give you the possibility to easily determine the absolute cell number of the subset of interest by "dual-platform" assay. Before any cytometric determination, I lyse RBC of a small aliquot of blood with 2% acetic acid in H2O (20 microliters of blood by reverse-pipetting and 180 of diluted acetic acid) and count at the hemocytometric chamber. This tells the number of nucleated cells (i.e of CD45+ cells, unless erythroblasts/CD45- are present), while staining in parallel with other mixes one tube with CD45 gives the measure of how the leukocyte gate drawn in a physical parameters plot is close to that drawn in a SSC vs CD45 (i.e. the quality of lyse).
Obviously, each operator has to validate this protocol in his/her lab by the use of count-beads or true-count tubes. Personally, I have validated this protocol in a side-by-side comparison against the ADVIA hematology system present in my institute for EDTA vacutainers. Not only the absolute number of total leukocytes closely correlated with the manual count, but also the relative (and absolute) number of major leukocyte subsets including lymphocytes, monocytes, neutrophils and eosinophils.
Thank you for that very comprehensive answer Mario. I've seen many different protocols from different mAb manufacturers, however I was unable to find a generic protocol for flow analysis as you have briefly discussed above. Would there be any complete protocol set which I can use for initial blood preparation to analysis including dual platform assay?
Hi Harindra, I'm attaching an original protocol for both single and dual-platform assay with practical suggestion. Let me know if is clear and if it works in your hands
"Why we use pre-enrichment of rare thymic subsets rather than to detect this populations immediately?" The answer is "To save the aquisition time to collect enough events coming from subsets of interest but not from dumps"
The same issue is in "whole blood staining" vs "lyse/wash or lyse/no wash" vs "pbmc isolation before staining".
Hi all.
Denis, you are right reasoning about the pre-enrichment of rare cells of interest, and I have no concerns about ficoll gradient separation per se, if prepared PBMC are to be used for downstream analysis or culturing. All in all, gradient centrifugation is a reliable method to have at your disposal tons of mononuclear cells at once, ready, for example, to be sorted magnetically or by FACS. Calculating that 1 ml of blood contains 2-3 millions of mononuclear cells, theoretically, in order to obtain 20-30 millions of PBMC by RBC lysis , 15x volumes of RBC lysing solution, about 150 ml, are to be used. this makes the lysis of RBC an impractical solution for a large number of cells, considering that safety and sterility issues impose to work into sterile cabinets.
However, coming back to the original question raised by Harindra, regarding B and T cell phenotyping, I think that the problem of rarity is not an issue, that the staining of pre-lysed whole blood offers an optimal middle road between ficoll density gradient separation and lyse/no wash procedures, and that the precise enumeration of cells of a given phenotype (i.e. their absolute number, not only their relative frequency) it can give offers the opportunity to infer important informations in the context of human infectious/inflammatory diseases.
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