Materials

1. Culture media: see Table 1. All the reagents were supplied from Biological Industries, Beth Haemek, Israel.

2. Cytokines, growth factors, antibodies: see Table 2.

Fig. 1. Electron micrograph of rat peritoneal mast cells (MC) co-cultured with Swiss

Albino 3T3 fibroblasts (FB) for 8 d (11).

Fig. 1. Electron micrograph of rat peritoneal mast cells (MC) co-cultured with Swiss

Albino 3T3 fibroblasts (FB) for 8 d (11).

Table 1

Culture Media

Culture media Supplements required

DMEM (4.5 g/L glucose) 10% v/v, FCS (heat inactivated)

100 U/mL penicillin 100 pg/mL streptomycin 2 mM L-glutamine

MEM-alpha (1 g/L glucose + l-glutamine) 10% v/v, FCS (heat inactivated)

100 U/mL penicillin 100 pg/mL streptomycin 10 pg/mL ribonucleosides/ deoxyribonucleosides

FCS Obtained from Biological Industries,

Beit Haemek, Israel

Trypsin-EDTA Obtained from Biological Industries,

Beit Haemek, Israel

3. Buffers: see Table 3.

4. Sensitive reagents: see Table 4. After the preparation of working solutions from these reagents, maintain them on ice in a foil-wrapped or blackened vessel. Whenever possible, prepare the working solution exactly in the required amount. The chromogenic substrates usually decompose with time. They should be stored at -70°C for longer periods, but it is preferable to prepare them fresh whenever possible.

Table 2

Cytokines, Growth Factors, Antibodies

Cytokines, Growth Factors, Antibodies

Table 2

Growth Factor/Antibody

Source"

Clone

Cat. No.

SCF

Amgen

(A generous gift)

AA1580-00

Anti-human, C. D.117

Pharmingen

YB5.B8

33651A

(c-kit)

Chimeric IgE Anti-NP

Serotec

JW8/1

MCA333B

Goat anti-mouse ^-chain

SouthernBiotech

Purified

1060-01

Anti-human, SCF

R&D

Purified

AF-255-NA

neutralizing antibody

"These were the sources for the reagents that were used to obtain most of the data described in this chapter.

Table 3

Buffers and Solutions

"These were the sources for the reagents that were used to obtain most of the data described in this chapter.

Table 3

Buffers and Solutions

BufferContent

Hank's Balanced Salt

Solution (HBSS) Tyrode's buffer

TG (Tyrode's gelatin buffer) TG++ (Tyrode's gelatin buffer, supplemented) HBA (HBSS, BSA, Azide)

Permeabilization/Blocking buffer for IF Washing buffer for IF Citrate/phosphate buffer for ß-hexosaminidase

137 mM NaCl, 2 mM KC1, 10 mM Na2HPO4, 1mM

KH2PO4, pH 7.4 1X and 10X obtained from Biological Industries,

Beth Haemek, Israel 137 mM NaCl, 12 mM NaHCO3, 5.5 mM l-glucose,

2 mM KCl, 0.3 mM Na2HPO4 0.1% w/v gelatin in Tyrode's buffer

1.8mM CaCl2, 0.9 mM MgCl2 in, TG buffer

0.1% w/v bovine serum albumine, 0.01% sodium azide in HBSS 0.1% w/v saponin, 10% w/v bovine serum albumine,

0.1% v/v human serum, 10 mM HEPES in HBA 0.1% w/v saponin, 10 mM HEPES in HBA 0.12 M citric acid, 0.14 M Na2HPO4, pH 4.5

5. Cellular stains: see Table 5.

6. P-Hexosaminidase substrate: dissolve 20 mg of substrate (p-NP ^-acetyl P-d-glucosaminide) in 4.5 mL of deionized water and 3 mL of substrate buffer (0.12 M citric acid, 0.14 M Na2HPO4, pH 4.5), mix well. This yields an 8 mM substrate solution. Keep foil-wrapped at 4°C.

Table 4

Sensitive Reagents

Sensitive reagents Source" Cat. No.

Table 4

Sensitive Reagents

Metrizamide

Sigma

M-3383

Compound 48/80

Sigma

C-2313

ß-hexosaminidase chromogenic substrate

(p-nitrophenyl-N-acetyl-ß-d-glucosaminide)

Sigma

N-9376

Tryptase chromogenic substrate

(N-p-tosyl-gly-pro-lys-p-nitroanilide)

Sigma

T-6140

Cathpesin G chromogenic substrate

(N- succinyl-ala-ala-pro-phe-p -nitroanilide)

Sigma

S-7388

OPT

Sigma

P-0657

Percoll

Sigma

P-1644

Collagenase

Sigma

C-0130

Hyaluronidase

Sigma

H-3506

DNase

Sigma

D-5025

NH4OH (ammonium hydroxide)

Frutatom, Israel

Trichloroacetic acid

Frutarom, Israel

NaOH (sodium hydroxide)

BDH

5553510

DMSO

Sigma

D-5879

"These are the sources for the reagents that were used to obtain most of the data described in this chapter.

"These are the sources for the reagents that were used to obtain most of the data described in this chapter.

Table 5 Cellular Stains

Stain

Content

Toluidine blue 0.07% w/v toluidine blue in 60% ethanol pH 3.5

Kimura's stain 0.03% w/v toluidine blue, 0.001% w/v light green, 1.4% w/v saponin dissolved in 137 mM NaCl, 2.7 mM KCl, pH 7.4 Methylene blue 1% w/v methylene blue in 0.1 N boric acid, pH 8.5

Trypan blue Commercially obtained from Sigma, cat. no. T-8154

7. Tryptase substrate: dissolve 10 mg of substrate (N-p-Tosyl-Gly-Pro-Lys p-NA) in 630 pL of dimethyl sulfoxide (DMSO), which will yield a substrate solution of 25 mM. Keep aluminum foil-wrapped in -20°C.

8. Cathepsin-G substrate: dissolve 5 mg of substrate (N-Succinyl-Ala-Ala-Pro-Phep-NA) in 200 pL of 0.1 M NH4OH. This yields a stock substrate solution of 40 mM. Keep aluminium foil-wrapped in -20°C.

Table 6

Several Sources of Mast Cells

Table 6

Several Sources of Mast Cells

Mast cell type

Source

Subheading/Chapter

Ref.

RPMC

Rat peritoneum

3.1.1

14

BMDMC

Mouse bone marrow

8

HMC-1 (line)

Human mast cell leukemia

Chapter 15

15

CBDMC

Human umbilical cord blood

Chapters 7,8,15

16

HLMC

Human lung

3.1.2

21

HSMC

Human skin

3.1.2

18

HIMC

Human intestines

3.1.2

20

HCMC

Human heart

3.1.2

17

HUMC

Human uterus

3.1.2

19

9. 10% trichloroacetic acid.

10. OPT solution: dissolve 5 mg of ophthalaldehyde (OPT) in 0.5 mL of methanol, yielding a 10 mg/mL solution. Keep foil-wrapped in -20°C.

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