IF (suspension cells)

This protocol has been tested on I.29µ+, primary B cells and plasma cells, multiple myeloma cell lines and primary multiple myeloma samples.

1. PREPARE MULTIWELL SLIDES FOR CELL ADHESION

Wash with ethanol 100% (squeeze) and let dry

Add a drop of poly-L-lysine, undiluted

Let 20’ RT

Aspirate (Poly-L-lysine can be recycled!!) from side, wash with dH₂

1. ADHERE CELLS

Spot 20.000-40.000 cells suspended in 20-50 µL cell culture medium / hole (in a 8-hole slide)

Incubate 30’ at 37°C

Wash once with PBS

1. FIX

Add a drop of formaldehyde 3,7 %, incubate 10’ RT

Wash once with PBS (can stop here: add a drop of PBS, keep at 4°C)

1. PERMEABILIZE

Triton X-100 0,1 % in PBS for 5’

Wash thoroughly in PBS

1. BLOCK ASPECIFICITY

PBS 1% FCS, incubate 30’ RT

Wash once with PBS

Dry thoroughly around holes with blotting paper

1. ADD 1ry Ab

In PBS 1% FCS, 20-25 µl/hole

Incubate 1 hr RT

Wash thoroughly in PBS

1. ADD 2ry Ab

In PBS 1% FCS

Ex: AlexaFluor GAM 488 (1:200, use gamma specific Ab if staining IgM secreting B cells) or GAR 546 (1:400) or GAR 647 (1:400)

Incubate 1 hr RT, in darkness

Wash thoroughly in PBS

1. STAIN NUCLEI

Add Hoechst 10 µg/ml (stock at 1mg/ml, 100x), incubate 10’

Wash thoroughly in PBS, then in dH₂0

1. COVER

Add Mowiol/DABCO, 1 drop/hole

Place glass cover, gently squeeze out mowiol in excess (avoid bubbles)

Leave RT, covered overnight

(in alternative: add glycerol 80%, place cover, seal with nail polisher, and look under microscope in ten minutes)