B cell purification

PRIMARY B CELLS PREPARATION FROM MOUSE SPLEEN AND INDUCTION WITH LPS

v Prepare the following reagents:

v dissolve LPS into RPMI (without antibiotics nor FCS) in order to prepare a 100X stock (2mg/ml to become 20ug/ml in the medium); the medium must be warmed to 37°C

v prepare ACK buffer (see protocol below)

v prepare PBS/BSA 5% (stock 10x, you would use 0.5%)

v prepare PBS/BSA 0.5% /EDTA 2mM

v prepare NEM 1M (stock 100x, you would use 10mM)

v prepare protease inhibitors cocktail (Roche is 50x)

v prepare lysis buffers (SDS, mild & NP40, see protocol below)

v wash tubes to smash spleens with EtOH (or isopropanol)

v prepare β-mercapto-ethanol 50mM (stock 1000x, you would use 50μM): the bottle is 14.3M

v prepare FACS solutions

v PUT IN ICE ALL THE REAGENTS (PBS, PBS/BSA..) AND WARM TO 37°C ACK & RPMI

Take spleens from mice

Smash each spleen by putting it in a 6wells plate well, pushing with a 15ml falcon cap: collect the cells by washing the well with PBS

Pass everything through a cell strainer

Spin 10’ at 500xg

Wash 1x in cold PBS

Resuspend in ACK lysis buffer (5ml/spleen)

ACK buffer:

- 150mM NH₄Cl (8g in 1 liter)

- 1mM KHCO₃ (0.1g in 1 liter)

- 0.1mM EDTA (0.037g in 1 liter)

- pH 7.2 – 7.4

Incubate 5’ – 7’ at rt, no more. Flick the tube from time to time

Add cold PBS, 10 – 20 times the volume (50 ml circa)

Remove clumps by passing everything through a cell strainer

Spin and discard the super. If red cells lysis gone well the pellet is white

Wash in cold PBS/0.5%BSA (+EDTA 2mM if required by Miltenyi kit): use about 20ml for the wash; as soon as you resuspend into the 20ml count the cells, and then spin at 500-400xg

LABELLING: beads + cells

Resuspend into the required buffer (0.5%BSA/PBS) and beads volume:see the B cell isolation kit, mouse - Miltenyi Biotec – (130-090-862) for the details

mix and incubate 15’ at 6-12 or 4°C on low agitation

COLUMNS PURIFICATION

Prepare MACS Separation LS Columns – Miltenyi Biotec – (130-042-401) with MACS MultiStand - Miltenyi Biotec – (130-042-303) and MidiMACS Separation Unit - Miltenyi Biotec – (130-042-302)

Wash the column with 3ml of buffer PBS/0.5%BSA/EDTA 2mM (see kit instructions)

Wash the cells twice with 10-20x the beads incubation volume

NB: in the negative selection you can centrifugate at 500xg, if you instead are interested in te complexes beads+cells you should slow down the speed -300xg- (positive selection)

Remove supernatant completely, eliminating well all the clumps that would block the column

resuspend into 500ul buffer PBS/0.5%BSA/EDTA 2mM each 10^8 total cells (check kit instructions)

let the suspension pass through the column

wash the column three times (with 3ml di PBS/0.5%BSA/EDTA 2mM) and keep everything comes out (initial 500ul + 9ml washing buffer)

spin at 500xg the 9.5ml

wash with PBS (without BSA) and spin again

if you make lysates at day 0, you must wash 3-4 times in normal PBS in order to remove BSA

just before plating, count cells and put in ice te cells needed for FACS, secretions and lysates

spin and resuspend the others in RPMI with LPS and b-mercapto to the desired concentration (usually 1-2*10⁶ cells/ml)

POSITIVE FRACTION ELUITION

If you want to elute the cells that stayed in the column you can detach the column from the magnetic support and wash it with 5ml of PBS/BSA 0.5%

MEDIUM:

- RPMI

- 50μM β-mercapto-ethanol

- mouse LPS (Sigma L2637) 20μg/ml fc, diluted in RPMI at 2 mg/ml, 100x stock

Buffer:

- PBS

- 0.5% BSA

NB = B LYMPHOCYTES ARE ABOUT THE 45-50% OF TOTAL SPLEEN CELLS, SO IF YOU START FROM 100*10⁶ CELLS TOT YOU WILL GET 50*10⁶.