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BrdU Incorporation to Measure Proliferation

By Joan Ross,2014-08-08 23:34
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BrdU Incorporation to Measure Proliferation

BrdU Incorporation to Measure Proliferation

Reagents:

    FITC BrdU Flow Kit (BD Pharmingen, Cat # 559619) Contains anti-BrdU FITC,

    Cytofix/Cytoperm buffer, Perm/Wash buffer (10x), Cytoperm plus buffer, BrdU (10 mg/mL), DNase, and 7-AAD

Many of the components can be obtained individually.

     Anti-BrdU FITC conjugated mAb set (BD Pharmingen, Cat # 556028). This set contains the

    anti-BrdU FITC mAb and the mIgG1 FITC isotype control. These Abs are prediluted and 20

    µl should be used per test.

     BrdU (Sigma, Cat # B5002-5G). The stock is stored at -20?C. Dissolve 0.5 g in 45 ml of

    dHO, add 5mL 10x PBS and filter sterilize with 22 µm filter. Dispense in 1mL aliquots and 2

    freeze at -80?C. Give 0.1 ml of this 10 mg/ml solution per mouse (1 mg/mouse). DNase (Sigma, Cat # D-4513). Resuspend the lyophilized DNase to a concentration of 1

    mg/ml in DPBS. Dispense in 300 µl aliquots and store at -80?C. Prior to use, add 0.7 ml of

    DPBS/tube and treat cells with 100 µl (30 µg)/sample.

     BD Cytofix/Cytoperm buffer (BD Pharmingen, Cat # 51-2090KZ)

     BD Perm/Wash buffer (10x) (BD Pharmingen, Cat # 51-2091KZ)

     BD Cytoperm plus buffer (BD Pharmingen, Cat # 51-2356KC)

    BrdU Administration: All mice receive 1 mg BrdU in 0.1 ml PBS via i.p. injection. Our typical long term BrdU pulse is daily administration of BrdU i.p. for 7 days prior to harvest. Alternatively, BrdU may be diluted to 0.8 mg/ml in the drinking water. This should be made fresh and changed daily. Pulsing periods longer than 14 days may be toxic and/or fatal. For short term/in situ proliferation, administer 1mg BrdU in 0.1 mL PBS via tail vein injection 4 h prior to harvest.

    Controls: Either 1) treat 1 mouse with PBS and use anti-BrdU Ab or 2) use the anti-BrdU isotype done on a mouse that received BrdU.

     6For tissues in which we count the cells (i.e., lungs, spleen, and lymph nodes) stain 2x10

    cells/sample.

    For TGs stain appropriately typically 1 TG (or equivalent) but possibly less or more depending on the time point being examined and whether they are pooled or individual. Be sure to pass TGs through FACS tube cell strainer.

     Wash cells with 1 ml of FACS buffer. Spin at 1400 rpm for 5 min at 4?C. Prepare Fc Block (BD Pharmingen, Cat # 553142) 1:50 in FACS buffer

    ; Dump sup. from wash and add 25 l per tube (All tubes receive Fc Block).

    ; Incubate 10 min on ice.

     Prepare surface stain mix. Add 25 l of mix per appropriate tube. For example, if you are

    doing an isotype for one of these surface stains then you can not use a mix for that but must

    add everything individually.

    ; The surface receptors to identify will often vary. Most times these will be at least anti-

    CD45 PerCP, anti-CD8; APC, and gB tetramer PE (NIAID Tetramer Facility). 498-505

    ; gB tetramer PE dilution will vary from batch to batch and be determined 498-505

    individually for each lot.

    ; Anti-CD45 PerCP (BD Pharmingen, Cat # 557235, clone 30-F11) 1:25 in FACS

    buffer

    ; Anti-CD8; APC (BD Pharmingen, Cat # 553035, clone 53-6.7) 1:25 in FACS buffer

    ; Add 25 l of this mix directly to appropriate tubes already containing Fc block. Therefore

    the final concentrations are: Fc Block 1:100, anti-CD45 PerCP 1:50, and anti-CD8

    APC 1:50.

    ; Incubate on ice for 1 h in the dark (1h incubation is only necessary if staining with MHC

    multimers).

     Wash 1X with 1 ml FACS buffer. Spin at 1400 rpm for 5 min at 4?C. Resuspend cell pellets with 100 l BD Cytofix/Cytoperm. Incubate for 20 min on ice in the

    dark.

     Wash 1X with 1ml 1X BD Perm/Wash. Spin at 1400 rpm for 5min at 4?C. Resuspend cells with 100 ul BD cyotperm plus. Incubate for 10 min on ice. Wash 1X with 1ml 1X BD Perm/Wash. Spin at 1400 rpm for 5min at 4?C. Resuspend cell pellets with 100 L BD Cytofix/Cytoperm. Incubate for 5 min on ice.

     Treatment with DNase to expose incorporated BrdU

    o Add 0.7 ml DPBS to the 0.3 ml aliquot stored at -80?C

    o Resuspend cells with 100 ul diluted DNase (30 µg)

    o Incubate 1 h at 37?C.

     Wash 1X with 1mL 1X BD Perm/Wash. Spin at 1400 rpm for 5 min at 4?C. Resuspend cells with 50 µl of perm/wash buffer containing diluted anti-BrdU FITC

    o If using the FITC BrdU Flow Kit then use 1:50 dilution.

    o If using the anti-BrdU Ab set then mix 20 µl prediluted Ab with 30 µl permwash buffer

    per sample.

    o Incubate 20 min at RT in the dark.

     Wash 1X with 1 ml 1X BD Perm/Wash. Spin at 1400 rpm for 5min at 4?C. Resuspend in 200µl FACS buffer.

    o Samples may be collected immediately or the next day. If collected on the next day seal

    tubes in paraffin and store at 4?C in the dark.

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