Sputum Sample Processing for Single Cell Isolation and Live Recovery for Single Cell RNA Sequencing

5mM EDTA in PBS

500ul 0.5M EDTA stock



49.5ml PBS. Store at 4C

10% BSA stock

Add 1g BSA to 10ml PBS, mix gently till dissolved



Filter sterilize & keep at 4C

50mM MgCl2 solution

Add 238mg of MgCl2 to 50ml of H2O 

10% DMSO/FCS freezing mixture

5mL DMSO

`45mL` fetal calf serum (FCS)

NALC in PBS 7.5Mass Percent NALC in PBS

DNase

Aliquoted into 100ul aliquots at 224kuml and stored at -20C

Add 100ul to 40ml dissociation buffer to make a final conc of 0.56ku/ml.

Collagenase IV

Add `500µL` to `40mL` dissociation buffer to make a final conc of 0.05mg/ml



 Aliquoted `500µL` at 4mg/ml and stored at -20C.

Make fresh for each use

1% BSA in PBS

5mL 10% BSA

`45mL` PBS

Dissociation buffer

1.6mL of50millimolar (mM) MgC₂2

`100µL` DNase



`500µL` collagenase

37.8mL PBS

Collect an induced sputum, preferably 5mL or more in volume, and process as below as soon as possible.

Weigh the sputum collection cup before and after the sputum collection.

Estimate the volume of sputum collected using a graduated container of the same size.

Record the viscosity (salivary, mucosalivary, purulent, mucopurulent)

Record the time of sputum collection

Record the time of sputum processing

Using a wide bore tip (or cut the bottom of a 1000µL) draw up ~200µL sputum. If the sample is too viscous for a pipette, use a syringe needle instead.

Add to a 1.5ml tube containing 500µL Zymo RNA Shield

Vortex tube for 0h 0m 10s

Immediately transfer to -80°C

Add 40mL dissociation buffer into the collection tube containing the sputum sample

Secure cap, manually invert the tube several times. Secure to a hula mixer (low setting) and incubate for 0h 10m 0s at Room temperature.

Note: Big clumps should dissolve after 0h 10m 0s. Some small fibers may will remain.

Add DTT to a final concentration of 1-2mM.

Invert the tube several times and incubate for 0h 5m 0s

Pass the dissociated sample through a 100 µm strainer on top of a new 50mL tube using an automated pipettor (do not pour). 350rcf,4°C

Wash the strainer with 2mL of PBS

Spin tube in a sealed bucket at 250rcf,4°C

Remove 1000µL supernatant and place in a cryovial for Luminex Cytokine assays. Add 140µL of protease inhibitor and store at -80°C

Aspirate the remainder of the supernatant and discard.

Gently resuspend cell pellet in 1mL EDTA/PBS using a wide bore 1000µL tip

Count cells using Turks protocol followed by paraformaldehyde fixation to decontaminate

Pre-cool CellCool and cryovials at 4°Cfor >2h 0m 0s

Place cells On ice

Gently mix the cells

Centrifuge at 300rcf,4°C

Discarded supernatant and resuspend cell pellets in pre-chilled (4°C) 10% DMSO/FCS solution (add dropwise)

Dispense cell suspension in 500µL aliquots into the pre-cooled cryovials and place into CoolCell at -80°C

Pilot samples

A	B	C	D
Sample	S1	S2	S3
Viscosity	mucosalivary	mucosalivary	mucosalivary
Volume used (ml)	10	4	6
Total cells before freezing (10^6)	2.2	0.9	2.9
Total cells recovered after thawing (2 weeks after frozen; 10^6)	0.88	0.48	1.33
Percentage cell recovery post freezing (%)	40	53	46

Clinical samples

A	B	C	D	E	F	G	H	I	J	K	L	M	N
Sample ID	Time to sputum processing (h.m)	weight of sputum (g)	volume of sputum (ml)	viscosity of sputum	Time to thaw (days)	Total cell count pre freeze (x10^6)	Total cell count taken forward pre freeze (x10^6)	Total cell count post freeze (x10^6)	Total cell count difference (x10^6)	Live cell count	Dead cell count	Viability (%)	Viability of prefreeze total (%)
CC20	2.85	17.8	15	salivary	662	9.1	7	4.86	2.14	1.58	3.28	33	23
CC22	<6.00	2.1	2	mucoid	658	1.3	1.3	0.60	0.71	0.15	0.44	26	12
CC23	2.10	2.9	3	mucosalivary	657	0.43	0.43	0.37	0.06	0.08	0.29	21	18
CC24	1.10	8.7	8	mucosalivary	657	1.1	1.1	0.81	0.30	0.19	0.62	23	17
CC25	4.90	0.7	0.9	mucosalivary	652	0.17	0.17	0.00	0.17	0.00	0.00	0	0
CC26	2.00	1.5	1.4	mucosalivary	652	1.13	1.13	0.73	0.40	0.17	0.57	23	15
CC27	2.20	3.2	3	mucosalivary	651	1.4	1.4	1.33	0.07	0.30	1.03	23	22
CC28	2.00	12	10	mucoid	644	3.2	2	2.74	-0.74	0.29	2.45	11	15
CC33	2.00	3	3	mucosalivary	627	0.8	0.8	0.67	0.13	0.25	0.42	37	31
CC37	<6.00	4.1	4	mucosalivary	623	0.92	0.92	0.97	-0.05	0.34	0.63	35	37