Isolation of BCells From Lymph Node Biopsies

1. Equipment: Sterile metal scissors and forceps, sterile small metal sieve, magnetic cell separator, and columns (MACS, Miltenyi Biotec, Bisley, UK). The specific separator and columns required would depend on the scale of the experiment; see manufacturer's instructions for details (http://www.miltenyibiotec. com). See Notes 1-3.

2. Preparation medium: RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 |g/mL penicillin G, 100 |g/mL streptomycin, 1 mM sodium pyruvate, 1% (v/v) nonessential amino acid solution; and 0.5 |g/mL Fungizone (all from GIBCO, Paisley, UK).

3. B-cell medium: IMDM medium supplemented with 10% (v/v) human serum (Sigma, Poole, UK), 2 mM L-glutamine, 20 |g/mL gentamycin, 100 |g/mL penicillin G, and 100 |g/mL streptomycin.

4. MACS buffer: phosphate-buffered saline (PBS; 125 mM NaCl, 16 mM Na2HPO4, 10 mM NaH2PO4, pH 7.2) supplemented with 10% (v/v) heat inactivated human serum, 0.5% (w/v) bovine serum albumin, and 2 mM ethylenediaminetetraacetic acid.

5. Ficoll-Paque Plus (Amersham Pharmacia Biotech, Little Chalfont, UK).

6. B-cell Isolation Kit (MACS, Miltenyi Biotec, Bisley, UK). The kit contains hap-ten-conjugated CD2, CD4, CD11b, CD16, CD36, and IgE antibodies (antibody cocktail); FcR-blocking reagent; and magnetic microbeads conjugated to a monoclonal antihapten antibody.

Table 1

Recommended Initial Conditions for Using Some Bcl-2 Family Protein Antibodies

Concentration

Table 1

Recommended Initial Conditions for Using Some Bcl-2 Family Protein Antibodies

Concentration

(Mg/mL)

Secondary antibody

Tertiary antibody

Approx Mw

Antibody

Source

or dilution3

(dilution)

(dilution)

Buffer used6

(kDa):

Rabbit anti-human

R&D Systems

0.5

Anti-rabbit HRP

n/ac

TBS

30

Bcl-XL

(AF800)

(1:5000)

Rabbit anti-human

Santa Cruz

Anti-rabbit HRP

n/a

TBS

42

Mcl-1

(S-19)

0.5

(1:5000)

Hamster anti-human

Pharmingen

Rabbit anti-hamster

Anti-rabbit HRP

TBS

26

Bcl-2

(clone 6C8)

0.5

(1:1000)

(1:5000)

Rabbit anti-human

Santa Cruz

0.4

Anti-rabbit HRP

n/a

TBS

21

Bax

(N-20)

(1:5000)

Goat anti-human

Santa Cruz

0.4

Anti-goat HRP

n/a

TBS

60

Bnip3

(C-18)

(1: 2000)

Rabbit anti-human

Sigma

1:500

Anti-rabbit HRP

n/a

TBST

42

Actin^

(20-33)

(1:5000)

aThe dilution factor used is given where the concentration is not known.

^Indicates whether blocking buffer contains TBS or TBST and whether TBS or TBST is used for washing the membrane. cn/a, not applicable.

^Analysis of actin expression is a useful way to confirm equivalent loading of intact proteins for each sample.

aThe dilution factor used is given where the concentration is not known.

^Indicates whether blocking buffer contains TBS or TBST and whether TBS or TBST is used for washing the membrane. cn/a, not applicable.

^Analysis of actin expression is a useful way to confirm equivalent loading of intact proteins for each sample.

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