Oct 10, 2021

Public workspaceWhole Cell Patch Clamp of Dispersed Human Islet Cells

DOI
dx.doi.org/10.17504/protocols.io.bv3un8nw
  • 1Department of Pharmacology, University of Alberta;
  • 2Alberta Diabetes Institute
  • Human Islet Research Network
Lili Liang
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DOI: dx.doi.org/10.17504/protocols.io.bv3un8nw
External link: https://hpap.pmacs.upenn.edu/explore/workflow/islet-physiology-studies?protocol=10
Protocol Citation: Xiaoqing Dai, Austin Bautista, Patrick E Macdonald 2021. Whole Cell Patch Clamp of Dispersed Human Islet Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bv3un8nw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 24, 2021
Last Modified: October 10, 2021
Protocol Integer ID: 51028
Keywords: HPAP, HIRN, Whole Cell Patch Clamp
Abstract
Cells use exocytosis to secrete a wide variety of molecules, including proteins, hormones, and neurotransmitters. Exocytosis can be monitored at the single-cell level by using patch-clamp electrophysiology to measure changes in membrane capacitance as vesicles fuse with the cell membrane and release their content. Dispersion of pancreatic islets into single cells allows for individual characterization of electrophysiological characteristics and allows for collection of cellular content for recovery of full-length transcriptomes by use of Smart-seq2.

Described in this protocol is the dispersion of pancreatic islets into single cells followed by whole-cell patch clamp electrophysiology which includes parameters representing cell size, exocytosis, sodium channel currents, and calcium channel currents. Cells are then collected individually after recording to be processed for single-cell RNA sequencing.


Note
Date Revised: September 9, 2020


Guidelines

References

CITATION
Joan Camunas-Soler, Xiao-Qing Dai, Yan Hang, Austin Bautista, James Lyon, Kunimasa Suzuki, Seung K Kim, Stephen R Quake, Patrick E MacDonald. Pancreas patch-seq links physiologic dysfunction in diabetes to single-cell transcriptomic phenotypes. Cell Metabolism, 2020.
LINK
https://doi.org/10.1016/j.cmet.2020.04.005

Materials
Equipment and recording solution

  1. EPC10 patch-clamp amplifier (HEKA Instruments Inc, Germany)
  2. Inverted microscope (Zeiss)
  3. Motorized micromanipulator (Sutter Instrument, MP-225)
  4. PatchMaster and Fitmaster Software (HEKA Instruments Inc, Germany)
  5. Capillary Glass tubing with flame polished ends pipettes (Warner Instrument)
  6. Extracelluar recording solution (in mM): 118 NaCl, 20 Tetraethylammonium, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 5 HEPES, and either 1, 5 or 10 glucose (pH 7.4 with NaOH)
  7. Pipette recording solution (in mM): 125 Cs-glutamate, 10 CsCl, 10 NaCl, 1 MgCl2, 0.05 EGTA, 5 HEPES, 0.1 cAMP, and 3 Mg-ATP (pH 7.15 with CsOH).
  8. Lysis buffer for collecting cells: H2O Amount1340 mL , recombination RNase inhibitor Amount50 mL , ERCC (1:600000) Amount50 mL , 10% Triton Amount10 mL , 10mM dNTP Amount500 mL , 100mM dT Amount50 mL , total Amount2000 mL .  dT is a customized oligo (IDT), the sequence is as follows:

AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TVN


Equipment
patch-clamp amplifier
NAME
EPC10
TYPE
HEKA
BRAND
895273
SKU
https://www.heka.com/products/products_main.html#physiol_patch
LINK

Equipment
Inverted microscope
NAME
Zeiss
BRAND
None
SKU
https://www.micro-shop.zeiss.com/en/us/system/Inverted+microscopes/1005
LINK

Equipment
Motorized micromanipulator
NAME
MP-225
TYPE
Sutter Instrument
BRAND
None
SKU
https://www.sutter.com/MICROMANIPULATION/mp225.html
LINK

Equipment
PatchMaster and Fitmaster Software
NAME
HEKA
BRAND
None
SKU
https://www.heka.com/downloads/downloads_main.html#down_patchmaster
LINK

Equipment
Capillary Glass tubing with flame polished ends pipettes
NAME
Warner Instrument
BRAND
None
SKU
https://www.warneronline.com/products/326
LINK








Procedure
Procedure
On the day receiving the shipped human pancreatic islets, hand-picked islets are dissociated to single cells using StemPro accutase (Gibco/Fisher, A11105-01). Plate cells in Thikness35 mm cell culture dishes, and culture in DMEM with 5.5 mM glucose, 10% FBS, and 100 U/mL penicillin/ streptomycin for 1-4 days.

Start patch clamping single cells after one overnight incubation, and continue patching cells for up to 4 days. Electrical activities are measured by using a pipette coated with sylgard (3 ~ 5 MΩ) in a heated chamber (32–35°C). Quality control is assessed by the stability of seal (>10 GΩ) and access resistance (<15 MΩ).
Perform electrical activity measurements in 1 minute from “break in” of cell membrane; measurement protocols include (in order): exocytosis, voltage-gated Na and Ca channel currents activated at -10 mV, and -120 mV, voltage-gated Na and Ca channel currents activated from -60 to +30 mV, steady-state inactivation of voltage-gated Na channel currents, reversal potential, hyperpolarization-activated non-selective cation currents activated at -140 mV.
When finishing all the measurements, use another big-tip pipette (0.2 ~ 0.5 MΩ) prefilled with Amount0.5 mL lysis mix, suck the cell into the pipette, and transfer it into a Amount0.2 mL PCR tube prefilled with Amount4 mL lysis mix.

Save cells in Temperature-80 °C freezer before shipping out for sequencing.

Data Analysis
Data Analysis
Using the software of Fitmaster (HEKA Instruments Inc, Germany), analysis is performed on the level of recording traces.
Citations
Joan Camunas-Soler, Xiao-Qing Dai, Yan Hang, Austin Bautista, James Lyon, Kunimasa Suzuki, Seung K Kim, Stephen R Quake, Patrick E MacDonald. Pancreas patch-seq links physiologic dysfunction in diabetes to single-cell transcriptomic phenotypes
https://doi.org/10.1016/j.cmet.2020.04.005