Flow cytometry

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Flow cytometry - text to be edited

MHC Dextramers are designed to detect rare occurrence of antigen-specific T cells. However, a successful staining depends on an appropriate experimental design. The General staining procedure should be applicable for most experiments if the MHC Dextramers are handled according to their specifications and the flow cytometer has been set up properly. Different MHC alleles and MHC Dextramer reagents carrying distinct peptides could have dissimilar staining characteristics. This is among others because they have various affinities for the T- cell receptor. Staining patterns also depend on the type of cell sample and there will be variations among different donors.

The notes below should help in optimising experiments with a particular MHC Dextramer

Analysis of results:

MHC Dextramer® positive cells are most easily viewed by gating on live lymphoid cells and then making a plot showing MHC Dextramer on the x-axis and anti-CD8 antibody on y-axis. It is very important to gate on live cells as dead cells can obscure the staining picture. The viability of the cell sample is recommended to be > 80%.

Controls:

Include a positive control if possible (e.g. specific T-cell clone/line, PBMC from a positive donor) and a negative control (T-cell clone/line negative for the used MHC Dextramer, PBMC from a negative donor, MHC Dextramer with another allele and/or irrelevant peptide).

Speeding up the staining procedure:

MHC Dextramers and conjugated anti-CD8 antibody can be mixed prior to addition to the lymphoid cells, then incubated at 4°C for 20 minutes followed by wash ect. (step 5-7 in the General staining procedure). Not all anti-CD8 antibodies can be used in this type of staining procedure. The clone SK1 (BD) recognizing human CD8 and clone KT15 or YTS169.4 (Dako) recognizing murine CD8 has been shown to work well together with the MHC Dextramers. Clone 53-6.7 (BD) cannot be used in this type of setup. It should be noted that mixing MHC Dextramer and conjugated anti-CD8 antibody prior to addition to the cells can result in a smaller signal to noise ratio compared to results obtained following the general staining procedure.

Unspecific staining:

Under certain conditions MHC Dextramer reagents can induce unspecific staining. Additional washing of the cells before and after MHC Dextramer staining can minimize such problem. If unspecific binding seems to be a problem it is recommended to compare different incubation times and temperatures or change pH of the incubation and washing buffers. Generally if the incubation temperature is lowered, increase the incubation time and if the incubation temperature is increased, shorten the incubation time. Even though 10 µl of MHC Dextramer is recommended per staining various MHC-peptide combinations can behave differently depending on e.g. cell material and affinity. Titration of the MHC Dextramer may help solve the problem.

Anti-CD8/CD3 antibodies:

Some anti-CD8 and anti-CD3 antibodies can interfere with MHC Dextramer binding. Be sure to use an antibody not interfering with MHC Dextramer binding. Titrate the anti-CD8 and/or anti-CD3 antibody before use. The clones SK1 (BD) recognizing human CD8, KT15/ YTS169.4 (Dako) recognizing murine CD8, SK7 (BD, eBiosciences)/ S4.1 (Invitrogen)/ UCHT1 (Dako) recognizing human CD3 all work well together with the MHC Dextramer.

Compensation:

When using flourochromes with overlapping emission spectra in the same sample remember to compensate the sample according to the specifications of the flow cytometer.

Fixation:

It is recommended to wash samples prior to fixation. Fixation should be carried out after staining with MHC Dextramers. Pre-staining fixation is not recommended.

1% methanol free formalin in PBS can be used as fixative following staining and washing. MHC Dextramers also work well with fixing and lysing reagents such as Uti-Lyse™ Erythrocyte-Lysing Reagent (Dako). Uti-Lyse™ lyses erythrocytes and fixes and stabilizes leucocytes in whole blood.

Intracellular staining:

Fixation and intracellular staining with Dako IntraStain, Fixation and Permeabilization Kit for Flow Cytometry can be used together with MHC Dextramers. Additional washing step prior to IntraStain Reagent A is essential for good results using MHC Dextramer™ together with the IntraStain procedure.

For research use only. Not for use in diagnostic procedures.

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