Jul 13, 2018
Version 7
Adult human small intestine cell dissociation (on ice) V.7
- 1Cincinnati Children's Hospital Medical Center
- Human Cell Atlas Method Development Community
Protocol Citation: Andrew Potter 2018. Adult human small intestine cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rnnd5de
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: July 13, 2018
Last Modified: July 13, 2018
Protocol Integer ID: 13742
Keywords: intestine, dissociation, single cell, CAP
Abstract
Protocol for human small intestine cell dissociation, performed on ice to reduce artifact gene expression.
Attachments
Guidelines
| Reagent | Storage Condition | |
| DPBS (Thermofisher, 14190144) | 4°C | |
| 0.5 M EDTA (Ambion, AM9260G) | room temp. | |
| BSA (Sigma, A8806) | 4°C | |
| Protease from Bacillus Licheniformis (Sigma, P5380) | Store 100 μL aliquots (100 mg/mL) in DPBS at -80°C |
Materials
MATERIALS
Please see Guidelines for required materials
Before start
Checklist prior to beginning:
-Centrifuges, large and small, set to 4 ºC
-Make enzyme stock; thaw two aliquots of Bacillus Licheniformis enzyme on ice.
-Make 0.04% BSA/PBS (50 mL)
-Things you need: petri dishes, clean forceps, razor blade, pipets, 30 μM filters, timer.
Stock solution for enzyme
895 μL DPBS
5 μL 0.5 M EDTA (2.5 mM final)
→Add 100 μL enzyme (100 mg/mL) to 900 μL of enzyme stock to make 1X enzyme mix. Add 28 mg of tissue to each 900 μL of enzyme mix.
While excluding as much PBS as possible, weigh out tissue using Mettler.
After weighing out tissue, transfer to petri dish on ice and mince tissue using grinding motion with razorblade for 2-3 minutes.
00:02:00 Mince tissue
After tissue is minced finely, add 1 mL enzyme mix per 28 mg of tissue to the petri dish and pipet minced tissue + enzyme into 1.5 mL tube (on ice).
1 mL enzyme mix per 28 mg of tissue
Start timer. Leave tube on ice - initially shake vigorously to break up the tissue, 3-
5x every 30-45 seconds for 5 minutes.
00:05:00
00:00:30
Now, when big chunks are broken up, shake every 1 minute while leaving on ice for 5 additional minutes (10 minutes total time).
00:05:00
00:01:00
After 10 minutes total digest time, triturate the digest mix 10X using p1000 set to 700 µL.
Continue shaking every minute for 5 additional minutes (15 minutes total time).
00:05:00
00:01:00
After 15 minutes digest time, triturate digest mix again 10X and spin digest mix at 90 G for 30 seconds at 4 °C.
00:00:30 Spinning
4 °C
Remove supernatant (80%) containing single cells and filter using 30 μM filter while leaving chucks on bottom; rinse filter with 10 mL PBS/BSA into 50 mL conical (on ice) to save single cells.
10 mL PBS/BSA
To residual chunks of tissue add additional 1 mL of enzyme (per 28 mg tissue).
1 mL enzyme (per 28 mg tissue)
Shake vigorously 3-4X every minute for 10 additional minutes (25 minutes total time).
00:10:00 Shaking.
00:01:00
Triturate again 10X using 1 mL pipet set to 700 µL.
Continue to shake vigorously every minute for 5 minutes additional time (30 minutes total time).
00:05:00
00:01:00
Triturate again 10X and filter using the same 30 μM filter and rinse with 10 mL PBS/BSA into the same 50 mL conical (on ice).
10 mL PBS/BSA
Divide flow-through into 2 15 mL tubes.
Spin 600 g for 5 minutes at 4 °C.
4 °C Spinning
00:05:00 Spinning
Carefully remove supernatant - re-suspend both pellets in 100 µL total PBS/BSA in one of the 15 mL conicals.
100 µL PBS/BSA
Add 700 μL RBC lysis buffer to 100 μL PBS/BSA (800 μL total). Triturate 20X using 1 mL pipet.
700 µL RBC lysis buffer
100 µL PBS/BSA
Incubate for 3 minutes on ice.
00:03:00 Incubation
Add 10 mL of PBS/BSA to 15 mL conical to dilute the RBC lysis buffer.
10 mL PBS/BSA
Spin 600 G for 5 minutes at 4 °C.
00:05:00 Spinning
4 °C
Remove supernatant.
Briefly re-suspend cells in a small volume of PBS/BSA and check to ensure that there are no more RBCs present.
Re-suspend in 10 mL total PBS/BSA in the same 15 mL conical.
10 mL PBS/BSA
Spin 600 g for 5 minutes at 4 °C.
4 °C Spinning
00:05:00 Spinning
Remove supernatant and re-suspend in a small volume of PBS/BSA to check cell concentration.
Analyze quantity and viability of cells using a hemocytometer with trypan blue: add 10 µL of trypan blue to 10 µL of cell suspension, mix by pipeting and pipet into hemocytometer; for Chromium, make concentration to 1 million cells per mL. For DropSeq, make concentration to 100,000 cells/mL.