TITLE: Human axillary lymph node fine-needle aspirate sample processing and cyropreservation
AUTHORS: Jacqueline HY Siu, cdendrou

[DESCRIPTION]
This protocol was used to generate a single-cell suspension from fine-needle aspirate samples of the adult human axillary lymph nodes and cyropreserved in CS10 for < 6 months. Cell yield was highly dependent on the participant; however, viability was >90% prior to freezing and between 80-90% after thawing (trypan blue dye exclusion).

[BEFORE_START]
Just prior to starting: 
Pre-cool centrifuge to 4ºC
Pre-cool cyropreservation vials in the -20ºC
Pre-cool cell freezing container if necessary 

Reagents: 
R10 Media is made up with RPMI 1640 with 25 mM HEPES (Cat No: R5886, Sigma), 10% heat inactivated fetal bovine serum (HI-FBS) (Cat No: F4135, Sigma), 1% Pen/Strep (Penicillin-Streptomycin 10,000 Units/mL (Cat No: 15140-122, Gibco), and 2 mM of L-Glutamine (Cat No: 25030-024, Gibco). Sterile bottle 500 mL filter system 0.22 μM (Cat No: 430758/ 430769 Corning or Cat No: SEGPU0538/ SEGPU0545, Merck) or 0.22 μM syringe filters. Store at 4ºC when not in use for a maximum of 1 month.  
Cell Wash Buffer is made up with PBS with 2% human AB serum and 2 mM EDTA. Store at 4ºC when not in use.  

[GUIDELINES]
All steps should be performed at 4ºC unless stated otherwise. 

[STEPS]
SECTION: Sample processing
2. Upon sample arrival, top up tubes with cold RPMI + 5% human AB serum (serum has been previously heat inactivated and sterile filtered) as required to ensure even volumes.
SECTION: Sample processing
3. Centrifuge at 400xg at 4ºC for 10 min. 400 x g, 10 min, 4 °C 

SECTION: Sample processing
4. Remove the supernatant, resuspend in 5 mL of red blood cell (ACK) lysis buffer (Gibco, Cat: A10492-01). Incubate at room temperature for 5 min (check colour), up to 10 min max. Top up with cell wash buffer (PBS + 2% FBS, 2 mM EDTA). Room temperature 

SECTION: Sample processing
5. Centrifuge at 400xg at 4ºC for 10 min. Wash again with cell wash buffer (PBS + 2% FBS, 2 mM EDTA). 400 x g, 10 min, 4 °C 

SECTION: Sample processing
6. Adjust final volume to 1 ml of cold RPMI + 5% AB serum. 
SECTION: Sample processing
7. Take a 10µl aliquot of the cell suspension and dilute with 10µl Trypan Blue. Load this mixture onto a haemocytometer and perform a cell count.
SECTION: Sample cyropreservation
8. Label the tubes and chill at -20ºC for ~ 10 minutes. The minimum number should be ~500,000 cells (to be frozen in 100 µl; 100 µl is the minimum freezing volume). Aliquot cells such that there is a maximum of ~1 million cells per cryovial.


SECTION: Sample cyropreservation
9. Add up to 10 ml of cold RPMI + 5% hAB serum to the tubes. Centrifuge at 400xg for 10 min at 4ºC.400 x g, 10 min, 4 °C 

SECTION: Sample cyropreservation
10. Resuspended samples in CS10 medium at a concentration of 1 ×106/100 µl. 
SECTION: Sample cyropreservation
11. Once fully mixed, aliquot 100 µl of the sample into the chilled cryotubes.
SECTION: Sample cyropreservation
12. Transfer the cryotubes into appropriate cell freezing container. Ensure all slots of the MrFrosty are filled, using the filler vials if necessary. 
SECTION: Sample cyropreservation
13. Transfer the samples into liquid nitrogen. Ideally this transfer should be performed within 24 hr, but may be extended to a maximum of 72 hr.   

SECTION: Sample collection &amp; transfer
1. Collect lymph node FNA samples in sterile filtered R10 media in 15 mL Falcon tubes. Transfer samples in an appropriate container and keep the tubes at 4ºC using cooled gel packs.