I'm attempting to patch clamp T-lymphocytes as part of a collaboration. I've been patch clamping for 10+ years in brain slices and cultured neurons / expression systems, but have run into difficulties when attempting to rupture the seal with CD4+ T-lymphocytes.
T-lymphocytes are isolated from spleen on the day of recording using a CD4+ T cell negative selection kit (StemCell #19752).
Recordings are performed on the same day within around 6 hours.
Solutions contain (mM):
Internal = K-Gluconate (120.0), HEPES (10.0), MgCl2.6H2O (1.0), CaCl2 (1.0), KCl (11.0), EGTA (11.0), Mg-ATP (4.0), Na-GTP (0.5)
External = NaCl (126.0), KCl (2.5), HEPES (10.0), CaCl2 (2.0), MgCl2 .6H2O (2.0), D-Glucose 10.0).
I find the cells seal very well in general and within around 5-10 seconds. However, when I apply suction the membrane seems very elastic and continues to be sucked into the pipette (pipette resistance = 4-6MOhm) until practically the entire cell is in the pipette bore. I've tried pipettes up to 8 MOhM which can help with the cell being sucked into the pipette, but the cells just seem to die rather that achieve whole-cell.
I think perhaps I'm just not recognizing when the patch has achieved whole-cell access and continuing to apply pressure once whole-cell, but i'm not sure I assume the transients are very small .
I resorted to using escin in perforated patch to try to figure out if I'm just not rupturing the seal at all, which gave whole-cell transients in the range of 25-50pA, but the cells seem to die fairly quickly with this approach.
Does anyone have any tips on undertaking patch clamp on T-lymphocytes? I saw a JoVE video on the process, but they didn't seem to have any issues going whole cell.
Thanks in advance.
We used gramicidin for periferal blood T-lymphocytes, but we were not isollating only CD4+. It worked well, and transients were about 200 pA.
You may try it.
Hi,
I used U-937 cells in the past, which is not the same but similar to the ones you work with. First of all, resistance of my electrode was 10 MOhm, that is bit higher than yours. In addition, you did not mention polishing of the pipette. I always polished them with a microforge. Finally, there is one more important question: who prepares the lymphocytes? In my past there was a half-year-long period when I could not patch clamp cultured neuronal cell lines. They looked nice in the microscope, they worked well in any assay, but patch clamping did not work because the cells always died when I was trying to rupture the cell membrane. Half a year later I found the reason: the glass flasks containing the culture media were washed with a special detergent proposed by the maker for culturing environments.... The detergent adhered to the wall of the glass and then diffused into the culture media. It did not disturb the cells, except that the cell membrane became weaker a little, making rupturing impossible. When I changed to single-use plastic flasks, this problem vanished. So, be aware of it!
Best, Imre
Hi, thanks for your answers.
Carmen - I will try gramicidin.
Imre - Yes we fire polish. Our collaborating lab were preparing the cells for me, so I guess there is a chance they are unwell. I'll go through the protocol with them.
Thanks once again.
Stuart
Are you sure that electrode needs to attach the cell examine as near as possilbe. One may need press the cell adherely down. Because lymphocyte is round in shape, small in size and floating, the experimenter needs to ensure that electrode is tighly attached to the cell before the suction was made.
we used poly-l lysine 0.01 % to attach the cells before patching them, I have seen some papers that use higher poly-l concentrations to attach the cells. Maybe this could helpjavascript:
Yes, this could be part of the issue. The cells were not attached to the coverslip. Although poly-d-lysine was used, they still moved when touched with the patch pipette.
I'll investigate the use of higher poly-d-lysine concentrations as well as physically "pinning" the cell using the pipette.
Many thanks.
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