Step-by-step lamina propria cell isolation protocol for H. polygyrus infected intestines

Step-by-step lamina propria cell isolation protocol for H. polygyrus infected intestines

according to Ferrer-Font et. al. 2019

Reagents

• RPMI (Gibco RPMI Medium 1640 #11875-093)
• HBSS (Gibco Hank’s Balanced Salt Solution #14175-095)
• DPBS (Gibco Dulbecco’s Phosphate Buffered Saline #14190-144)
• UltraPure 0.5M EDTA (Thermofisher #15575020)
• DNAse I (Roche #10104159001, 2916 Kunitz units/mgL)
• Collagenase A from Clostridium histolyticum (Roche #10103578001, 0.223 U/mg solid)
• Fetal Bovine Serum (Gibco)
• FACS buffer (PBS, 2% FBS, 2mM EDTA) 
• EDTA wash buffer (HBSS, 2mM EDTA)
• Wash buffer (HBSS)
• Collection buffer (HBSS, 2% FBS)
• Digestion mix (RPMI, 20% FBS, 1mg/ml Collagenase A, 0.05mg/ml DNAse)
• Trypan Blue Solution (Gibco)

Materials

• Funnels
• 10x10cm Gauze (140µm mesh size)
• Tweezers to hold gauze in place
• 50ml falcon tubes
• 40µm cell strainers
• 100µm cell strainer
• 25 ml serological pipette
• Scissor/tweezers for collection
• Petri dish
• Haemocytometer

Equipment

• Shaking incubator
• Centrifuge

Step-by-step protocol

1. Warm bottles of RPMI, HBSS and HBSS/2mM EDTA to 37 °C.
2. Prepare 50ml falcon tubes with 10ml HBSS/2% FBS for each sample (keep on ice). Take petri dish, PBS and scissors/tweezers for collection of samples. 
3. Sacrifice mouse, spray with EtOH and perform midline incision of the skin and muscle layer (Appendix 1-figure 1A).
4. Expose the intestines and excise the entire small intestine or a segment of interest and separate it from mesentery/fat tissue (Appendix 1-figure 1B-D).
5. Place intestinal segment on moist paper and remove the Peyer's patches.
6. Cut the segment longitudinally (Appendix 1-figure 1 E).
7. Remove worms and intestinal content (Appendix 1-figure 1F&G).
8. Wash intestinal segment in PBS and cut into small pieces (~5mm long) (Appendix 1-figure 1H&I).
9. Collect pieces in 50ml falcon tube containing 10 ml HBSS/FBS, shake well and keep on ice (Appendix 1-figure 1J).
10. Repeat steps 3-8 for remaining samples.
11. Prepare 10ml fresh digestion mix for each sample containing 20% FBS, 1mg/ml Collagenase A and 0.05mg/ml DNAse in RPMI and warm up to 37°C.
12. Filter each sample through a 10x10cm gauze (140µm mesh size) placed on top of a funnel. Discard the flow-through (Appendix 1-figure 1K).
    The same gauze can be reused in each wash step.
13. Wash sample with 10ml of warm HBSS twice. Discard the flow-through (Appendix 1-figure 1l).
14. Remove the gauze from the funnel and collect sample in a 50ml falcon tube containing 10 ml HBSS/EDTA (Appendix 1-figure 1M-O).
    It is advisable to process no more than 4-5 samples in parallel.
15. Incubate samples for 10 minutes at 37° C and 200rpm in a shaking incubator (Appendix 1-figure 1P). 
16. Pulse vortex samples three times at 2500 rpm (maximum speed) for 3 seconds after each incubation.
    A cloudy suspension should be observed.
17. Repeat wash steps 12-16 two more times, for a total of three 10-minute wash steps. 
    The HBSS/EDTA suspension should become less cloudy with each wash step. If significant amounts of debris are still observed after the last wash add a forth wash step.
18. After the final EDTA wash step, filter each sample through a 10x10cm gauze and wash with 10ml of warm HBSS twice.
19. Remove the gauze from the funnel and collect sample in a 50ml falcon tube containing 10ml digestion mix.
20. Incubate samples for 30 minutes at 37 °C and 200rpm in a shaking incubator. Shake samples vigorously every 5 minutes.
    The pieces will not be fully digested at this time point but have been optimised to yield the highest cell number with the least effect on viability and epitope integrity. 
21. Add 10ml of FACS buffer to each sample and keep on ice to stop the digestion.
22. Filter each sample through a 100µm and 40µm cell strainer into a new 50ml falcon tube using a 25ml serological pipette and place on ice (Appendix 1-figure 1Q&R).
    Do not simply pipette the sample on top of the strainer but force it through the mesh with pressure from the pipette controller.
23. Centrifuge the single cell suspension at 600 x g for 6 min at 4 °C.
24. Discard the supernatant and resuspend in 1ml of 20µg/ml DNAse containing FACS buffer.
    Use DNAse containing FACS buffer in all downstream procedures to reduce clumping.
25. Count live cells in a 1:1 suspension of Trypan Blue using a haemocytometer.
26. Process desired number of cells for flow cytometry or other downstream applications.
    Lamina propria digests contain a high percentage of cell debris, which will stain with viability dyes. Optimise the concentration of viability dye and record samples using a high Scatter threshold to facilitate the identification of cells. If cells are to be sorted, pre-enrich the samples by pre-sorting CD45+ cells or using positive selection beads. Avoid enrichment systems that are easily clogged.

Category